Multi-primer amplification method for barcoding of target nucleic acids

ABSTRACT

In certain embodiments, the present invention provides amplification methods in which nucleotide tag(s) and, optionally, a barcode nucleotide sequence are added to target nucleotide sequences. In other embodiments, the present invention provides a microfluidic device that includes a plurality of first input lines and a plurality of second input lines. The microfluidic device also includes a plurality of sets of first chambers and a plurality of sets of second chambers. Each set of first chambers is in fluid communication with one of the plurality of first input lines. Each set of second chambers is in fluid communication with one of the plurality of second input lines. The microfluidic device further includes a plurality of first pump elements in fluid communication with a first portion of the plurality of second input lines and a plurality of second pump elements in fluid communication with a second portion of the plurality of second input lines.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 14/180,262,filed Feb. 13, 2014, which is a division of U.S. application Ser. No.12/753,703, filed Apr. 2, 2010, U.S. Pat. No. 8,691,509, which claimsthe benefit of prior U.S. provisional application No. 61/166,181, filedApr. 2, 2009; prior U.S. provisional application No. 61/166,105, filedApr. 2, 2009; prior U.S. provisional application No. 61/186,327, filedJun. 11, 2009; and prior U.S. provisional application No. 61/305,907,filed Feb. 18, 2010, which are all hereby incorporated by reference intheir entireties.

FIELD OF THE INVENTION

The present invention relates generally to the area of high-throughputassays for detection and/or sequencing of particular target nucleicacids. In certain embodiments, the present invention providesamplification methods in which nucleotide tag(s) and a barcodenucleotide sequence are added to target nucleotide sequences.

BACKGROUND OF THE INVENTION

The ability to detect specific nucleic acid sequences in a sample hasresulted in new approaches in diagnostic and predictive medicine,environmental, food and agricultural monitoring, molecular biologyresearch, and many other fields. In addition, new sequencingmethodologies provide the means for rapid high-throughput nucleic acidsequencing.

Additional methods, especially methods that facilitate analysis of manytargets and/or the analysis of many samples simultaneously across abroad range of concentrations in a sample would be of great benefit.

Microfluidic devices can be used for analytical, preparative, metering,and other manipulative functions on a scale not imagined until recently.The advantages of microfluidic devices include conservation of preciousreagents and samples, high density and throughput of sample analysis orsynthesis, fluidic precision and accuracy at a level scarcely visible tothe unaided eye, and a space reduction accompanying the replacement ofcounterpart equipment operating at the macrofluidic scale. Associatedwith the reduction in size and the increased density of microfluidicdevices is increased complexity and higher engineering and fabricationcosts associated with increasingly intricate device architecture.

Recently, there have been concerted efforts to develop and manufacturemicrofluidic systems to perform various chemical and biochemicalanalyses and syntheses. Additionally, microfluidic devices have thepotential to be adapted for use with automated systems, therebyproviding the additional benefits of further cost reductions anddecreased operator errors because of the reduction in human involvement.Microfluidic devices have been proposed for use in a variety ofapplications, including, for instance, capillary electrophoresis, gaschromatography, and cell separations.

However, realization of these benefits has often been thwarted becauseof various complications associated with the microfluidic devices thathave thus far been manufactured. For instance, many of the currentmicrofluidic devices are manufactured from silica-based substrates,which are difficult and complicated to machine. As a result, manydevices made from such materials are fragile. Furthermore, transport offluid through many existing microfluidic devices requires regulation ofcomplicated electrical fields to transport fluids in a controlledfashion through the device.

Thus, in view of the foregoing benefits that can be achieved withmicrofluidic devices but the current limitations of existing devices,there remains a need for microfluidic devices designed for use inconducting a variety of chemical and biochemical analyses. Because ofits importance in modern biochemistry, there is a particular need fordevices that can be utilized to conduct a variety of nucleic acidamplification reactions, while having sufficient versatility for use inother types of analyses as well.

Devices with the ability to conduct nucleic acid amplifications wouldhave diverse utilities. For example, such devices could be used as ananalytical tool to determine whether a particular target nucleic acid ofinterest is present or absent in a sample. Thus, the devices could beutilized to test for the presence of particular pathogens (e.g.,viruses, bacteria, or fungi), and for identification purposes (e.g.,paternity and forensic applications). Such devices could also beutilized to detect or characterize specific nucleic acids previouslycorrelated with particular diseases or genetic disorders. When used asanalytical tools, the devices could also be utilized to conductgenotyping analyses and gene expression analyses (e.g., differentialgene expression studies). Alternatively, the devices can be used in apreparative fashion to amplify sufficient nucleic acid for furtheranalysis such as sequencing of amplified product, cell-typing, DNAfingerprinting, and the like. Amplified products can also be used invarious genetic engineering applications, such as insertion into avector that can then be used to transform cells for the production of adesired protein product.

Despite these advances in microfluidic design and use, it would beuseful to reduce the complexity of microfluidic chips and simplify theiroperation. Additionally, a need exists for an increased ability torecover reaction products from microfluidic devices. Thus, there is aneed in the art for improved methods and systems related to microfluidicdevices.

SUMMARY OF THE INVENTION

In certain embodiments, the invention provides a method for amplifying,tagging, and barcoding a plurality of target nucleic acids in aplurality of samples. The method entails preparing an amplificationmixture for each target nucleic acid. Each amplification mixtureincludes:

a forward primer comprising a target-specific portion;

a reverse primer comprising a target-specific portion, wherein theforward primer additionally comprises a first nucleotide tag and/or thereverse primer additionally comprises a second nucleotide tag; and

at least one barcode primer including a barcode nucleotide sequence anda first and/or second nucleotide tag-specific portion, wherein thebarcode primer is in excess of the forward and/or reverse primer(s).

Each amplification mixture is subjected to amplification to produce aplurality of target amplicons, wherein each target amplicon includes atagged target nucleotide sequence, with first and/or second nucleotidetags flanking the target nucleotide sequence, and at least one barcodenucleotide sequence at the 5′ or 3′ end of the target amplicon.

In specific embodiments, the forward primer additionally includes afirst nucleotide tag. If desired, the reverse primer can additionallyinclude a second nucleotide tag.

In certain embodiments of the tagging/barcoding method, theconcentration of the barcode primer in the amplification mixtures is atleast 4-fold the concentration of the forward and/or reverse primer(s).In variations of such embodiments, the concentration of the barcodeprimer in the amplification mixtures is at least 50-fold theconcentration of the forward and/or reverse primer(s).

In particular embodiments of the barcoding/tagging method, the firstand/or second nucleotide tags and/or the barcode nucleotide sequence areselected so as to avoid substantial annealing to the target nucleicacids. In illustrative embodiments, the barcode nucleotide sequenceidentifies a particular sample. Where the barcode primer includes abarcode nucleotide sequence and a first nucleotide tag-specific portion,in certain embodiments, a plurality of forward primers include the samefirst nucleotide tag. For, example, where multiple targets are to beamplified in different samples, the set of forward primers correspondingto the set of targets can all have the same first nucleotide tag.

In particular embodiments of the barcoding/tagging method, the forwardand reverse primers for each target are initially combined separatelyfrom the sample, and each barcode primer is initially combined with itscorresponding sample. For example, where T targets are to be amplifiedin S samples, T and S being integers greater than one, the method canadditionally include preparing S×T amplification mixtures wherein theinitially combined forward and reverse primers are added to theinitially combined samples and barcode primers.

In certain embodiments of the barcoding/tagging method, theamplification is carried out for at least 3 cycles to introduce thefirst and second nucleotide tags and the barcode nucleotide sequence. Invariations of these embodiments, the amplification is carried out forbetween 5 and 50 cycles. In particular embodiments, the amplification iscarried out for a sufficient number of cycles to normalize targetamplicon copy number across targets and across samples.

In certain embodiments of the barcoding/tagging method, at least 50percent of the target amplicons produced upon amplification are presentat greater than 50 percent of the average number of copies of targetamplicons and less than 2-fold the average number of copies of targetamplicons.

In other embodiments, the invention provides a method in which barcodingis, optionally, omitted and the target nucleotide sequences are taggedafter amplification. This method entails amplifying a plurality oftarget nucleic acids, typically, in a plurality of samples. Anamplification mixture is prepared for each target nucleic acid, whereineach amplification mixture includes:

a forward primer including a target-specific sequence; and

a reverse primer including a target-specific sequence;

Each amplification mixture is subjected to amplification to produce aplurality of target nucleotide sequences. The target nucleotidesequences are then tagged (e.g., by ligation of nucleotide tags unto oneor both ends of the target nucleotide sequences) to produce a pluralityof target amplicons. Each target amplicon includes first and/or secondnucleotide tags flanking the target nucleotide sequence. In particularembodiments, at least 50 percent of the target amplicons are present atgreater than 50 percent of the average number of copies of targetamplicons and less than 2-fold the average number of copies of targetamplicons.

In certain embodiments of the amplification methods described herein, atleast 70 percent of the target amplicons are present at greater than 50percent of the average number of copies of target amplicons and lessthan 2-fold the average number of copies of target amplicons. Inillustrative embodiments, at least 90 percent of the target ampliconsare present at greater than 50 percent of the average number of copiesof target amplicons and less than 2-fold the average number of copies oftarget amplicons.

In various embodiments, the average length of the target amplicons is atleast 25 bases, 50 bases, 100 bases, 200 bases, 500 bases, and 750bases. Longer average lengths, such as 1 kilobase or more are alsopossible, as, for example, when amplification is carried out bylong-range PCR. In such embodiments, amplification may yield targetamplicons wherein at least 70 percent of the target amplicons arepresent at greater than 50 percent of the average number of copies oftarget amplicons and less than 2-fold the average number of copies oftarget amplicons.

An advantage of the methods described herein is that amplification can(but need not) be carried out in small reaction volumes. In particularembodiments, the volume of the amplification mixtures is in the range ofabout 1 picoliter to about 50 nanoliters. In certain embodiments, thevolume of the amplification mixtures is in the range of about 5picoliters to about 25 nanoliters.

The methods described herein can, optionally, include recovering thetarget amplicons from the amplification mixtures. In certainembodiments, the target amplicons are recovered in a volume and/or copynumber that varies less than about 50% among the recovered targetamplicons. The recovered amplicons can be employed for furtheramplification and/or analysis (e.g., DNA sequencing). In someembodiments, at least one target amplicon can be subjected toamplification using primers specific for the first and second nucleotidetags to produce a target amplicon lacking the barcode nucleotidesequence, if such is desired.

In particular embodiments of the methods described herein, the targetnucleic acids include genomic DNA. In variations of these embodiments,the genomic DNA can be DNA intended for DNA sequencing, e.g., automatedDNA sequencing

In certain embodiments, one or more of the forward primer, reverseprimer, and barcode primer can include at least one additional primerbinding site. For example, if a barcode primer is employed, the barcodeprimer can include at least a first additional primer binding siteupstream of the barcode nucleotide sequence, which is upstream of thefirst nucleotide tag. In such an embodiment, the reverse primer caninclude at least a second additional primer binding site downstream ofthe second nucleotide tag. In particular embodiments, where the targetnucleotide sequences are to be sequenced by automated DNA sequencing,the first and second additional primer binding sites are capable ofbeing bound by DNA sequencing primers.

If a barcode primer is not employed, and the target nucleotide sequencesare tagged after amplification, the first and second nucleotide tags canbe capable of being bound by DNA sequencing primers.

Thus, the methods described herein can, optionally, include subjectingat least one target amplicon to DNA sequencing.

In certain embodiments, the method can, optionally, include quantifyingthe amount of target amplicons in the amplification mixtures. This stepmay be carried out, for example, prior to automated DNA sequencing. Inparticular embodiments, quantification includes recovering the targetamplicons and subjecting them to digital amplification. Digitalamplification includes, in particular embodiments,

distributing the preamplified target amplicons into discrete reactionmixtures, wherein each reaction mixture, on average, includes no morethan one amplicon per reaction mixture; and

subjecting the reaction mixtures to amplification.

Quantification in digital amplification may be carried out by real-timePCR and/or endpoint PCR.

The amplification methods described herein can, optionally, includedetermining the amount of each target nucleic acid present in eachsample. In certain embodiments, the methods can be performed indetermining the copy numbers of the target nucleic acids in each sample.In particular embodiments, the methods can be performed in determiningthe genotypes at loci corresponding to the target nucleic acids. Inother embodiments, the methods can be performed in determining theexpression levels of the target nucleic acids.

In particular embodiments, the present invention relates to microfluidicdevices. More particularly, the present invention relates to amicrofluidic device that provides for recovery of reaction products.Merely by way of example, the method and apparatus has been applied to aPCR sample preparation system used to prepare libraries for nextgeneration sequencing. However, it would be recognized that theinvention has a much broader range of applicability.

According to an embodiment of the present invention, a microfluidicdevice is provided. The microfluidic device includes a plurality offirst input lines and a plurality of second input lines. Themicrofluidic device also includes a plurality of sets of first chambersand a plurality of sets of second chambers. Each set of first chambersis in fluid communication with one of the plurality of first input linesand each set of second chambers is in fluid communication with one ofthe plurality of second input lines. The microfluidic device furtherincludes a plurality of first pump elements in fluid communication witha first portion of the plurality of second input lines and a pluralityof second pump elements in fluid communication with a second portion ofthe plurality of second input lines.

According to another embodiment of the present invention, a method ofoperating a microfluidic device having an assay chamber, a samplechamber, and a harvesting port is provided. The method includes closinga fluid line between the assay chamber and the sample chamber, flowing asample into the sample chamber via a sample input line, and flowing anassay into the assay chamber via an assay input line. The method alsoincludes opening the fluid line between the assay chamber and the samplechamber, combining at least a portion of the sample and at least aportion of the assay to form a mixture, and reacting the mixture to forma reaction product. The method further includes closing the fluid linebetween the assay chamber and the sample chamber, flowing a harvestingreagent from the harvesting port to the sample chamber, and removing thereaction product from the microfluidic device.

According to a particular embodiment of the present invention, a methodof preparing reaction products is provided. The method includesproviding M samples and providing N assays. The method also includesmixing the M samples and N assays to form M×N pairwise combinations.Each of the M×N pairwise combinations are contained in a closed volume.The method further includes forming M×N reaction products from the M×Npairwise combinations and recovering the M×N reaction products.

Many benefits are achieved by way of the present invention overconventional techniques. For example, embodiments of the presentinvention provide for mixing and reaction of M×N samples and assaysfollowed by recovery of the reaction products in sample-by-sample pools.Additionally, dilation pumping is utilized to remove substantially allof the reaction products from the microfluidic device, providinguniformity between the various reaction product pools. Utilizing thesystems and methods described herein, the time and labor required toprepare libraries is reduced in comparison with conventional techniques.These and other embodiments of the invention along with many of itsadvantages and features are described in more detail in conjunction withthe text below and attached figures.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be understood by reference to the followingdescription taken in conjunction with the accompanying drawings thatillustrate certain specific embodiments of the present invention.

FIG. 1 depicts an illustrative matrix-type microfluidic device in planview.

FIG. 2 is a simplified perspective illustration of a carrier and amicrofluidic device that permits recovery of reaction products.

FIG. 3 is a simplified schematic diagram of a microfluidic device thatpermits recovery of reaction products.

FIG. 4 is a simplified schematic diagram of several unit cells of themicrofluidic device illustrated in FIG. 3.

FIG. 5A is simplified schematic diagram of a microfluidic device thatpermits recovery of reaction products.

FIG. 5B is a simplified schematic diagram of portions of themicrofluidic device illustrated in FIG. 5A.

FIG. 6 is a simplified schematic diagram of several unit cells of themicrofluidic device illustrated in FIG. 5A.

FIG. 7 is a simplified flowchart of a method of operating a microfluidicdevice that permits recovery of reaction products.

FIGS. 8A-8D are simplified schematic diagrams illustrating fluid flowthrough unit cells of a microfluidic device that permits recovery ofreaction products during operation.

FIGS. 9A-9D are simplified schematic diagrams illustrating fluid flowthrough a microfluidic device that permits recovery of reaction productsduring operation.

FIG. 10 illustrates an embodiment of a 3-primer amplification method forbarcoding target nucleic acids prior to sequencing.

FIG. 11 shows a photograph of the gel described in Example 1. The lanesare as follows: (2) molecular markers, (4) sample amplified with 454tails; (5) sample (NTC) amplified with 454 tails; (7) sample amplifiedwith A5 primer pair; (8) sample (NTC) amplified with A5 primer pair;(10) sample amplified with 3 primers; and (11) sample (NTC) amplifiedwith 3 primers.

FIG. 12 shows gel-view electropherograms obtained from 4 Agilent 1KBiolanalyzer chips for each of the individual samples run on the AccessArray IFC (Integrated Fluidic Circuit) in Example 4. Each column in thefigure shows the size distribution of DNA products in each sample. Allsamples produce similar distributions of products

FIG. 13A-13B shows results from Example 4. A) Predicted sizes of all PCRproducts for this set of target specific primers. B) Electropherogram ofone of the sample pools obtained from the Access Array IFC. Distributionof product size within a single product pool. All products fall withinthe predicted size range shown in (B).

FIG. 14A-14C shows results from Example 4. A) Number of sequencescounted per barcode on the 454 sequence run. Upper horizontal linerepresents 2× average number of counts per barcode. Lower horizontalline represents 50% of average number of counts per barcode. B) Numberof sequences counted per amplicon. Each point on the plot represents thenumber of times the sequence for an individual chamber on the AccessArray IFC were measured on the sequencer. Triangular points representPCR reactions with greater than 2× the average representation. Dark greypoints represent PCR reactions with less than 0.5× the averagerepresentation. C) Frequency distribution of amplicon representation.The dark grey line represents the number of amplicons present at a givenrepresentation. The light grey line represents the number of reads thatwould be measured at a given coverage (e.g. 98% at 20× coverage).Percentage of amplicons within 2-fold of average: 95.8%; percentage ofamplicons within 5-fold of average: 99.7%.

FIG. 15A-15B shows an example of a multi-primer reaction set-up using 4outer primers with different combinations of primer binding site andnucleotide tags. (Example 5.) A) Two forward barcode primers(454B-BC-Tag8, 454A-BC-Tag8 and two reverse barcode primers(454A-BC-Tag5, 454B-BC-Tag8) are combined with one inner primer pair(Tag8-TSF and Tag5-TSR). B) The two major PCR products formed from thisPCR reaction. PCR products containing 454-A Tag8 and 454A-Tag5 at eachend or 454B-Tag8 and 454B-Tag5 at each end do not produce significantPCR products due to PCR suppression

FIG. 16A-16B shows the representation of each of the primer sequences ineach of the samples for each of the amplicons in FIG. 15B. The number ofsequences counted per amplicon were normalized to the average number ofcounts per amplicon within a sample. The normalized counts for anindividual amplicon were summed between the A and B emulsions (A) forTag5 amplicons in Emulsion A plus Tag 8 amplicons in Emulsion B and (B)for Tag5 amplicons in Emulsion B plus Tag 8 amplicons in Emulsion A. Themiddle dark grey line represents the average representation of eachamplicon. The upper light grey line represents 2× average coverage. Thelower light grey line represents 50% of average representation.

FIG. 17 shows the results from Example 6: Successful amplication of aPCR product using the 4-primer strategy designed for use on the IlluminaGA II sequencer. The barcode primers listed in Table 14 are labelled asOuter Short.

FIG. 18 shows results from Example 8: PCR reactions of three pools of 10sets of PCR primers (A, B, C) when the PCR reactions were run fortemplate-specific primers only and in 4-primer mode. The presence ofhigher molecular weight products in the 4-primer strategy demonstratessuccessful 4-primer assembly.

FIG. 19 shows results from Example 8: Changing the ratio of inner andouter primers impacts yield in multiplex 4-primer PCR using inner andouter primers.

DETAILED DESCRIPTION

In certain embodiments, the present invention provides amplificationmethods in which nucleotide tag(s) and a barcode nucleotide sequence areadded to target nucleotide sequences. The added sequences can then serveas primer and/or probe-binding sites. The barcode nucleotide sequencecan encode information, such as, e.g., sample origin, about the targetnucleotide sequence to which it is attached. Tagging and/or barcodingtarget nucleotide sequences can increase the number of samples that canbe analyzed for one or multiple targets in a single assay, whileminimizing increases in assay cost. The methods are particularlywell-suited for increasing the efficiency of assays performed onmicrofluidic devices.

In particular embodiments, the methods are used to prepare nucleic acidsfor DNA sequencing by, e.g., adding binding sites for DNA sequencingprimers, optionally followed by sample calibration for DNA sequencing.In specific, illustrative embodiments, the method can be employed to addbinding sites for DNA sequencing primers in a microfluidic device thatpermits recovery of reaction products. In illustrative devices of thistype, dilation pumping can utilized to remove substantially all of thereaction products from the microfluidic device, providing uniformitybetween the various reaction product pools. Thus, it is possible toproduce pools of barcoded reaction products that are uniform withrespect to volume and copy number. In various embodiments, the volumeand/or copy number uniformity is such that the variability, with respectto volume and/or copy number, of each pool recovered from the device isless than about 100 percent, less than about 90 percent, less than about80 percent, less than about 70 percent, less than about 60 percent, lessthan about 50 percent, less than about 40 percent, less than about 30percent, less than about 20 percent, less than about 17 percent, or lessthan about 15, 12, 10, 9, 8, 7, 6, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, or0.5 percent. Those of skill in the art appreciate that the volume and/orcopy number variability may fall within any range bounded by any ofthese values (e.g., about 2 to about 7 percent). In an illustrativeembodiment, the volume samples recovered from a microfluidic device varyby no more than approximately 10%. Standard pipetting error is on theorder of between 5 and 10%. Thus, the observed variability in volumes islargely attributable to pipetting error. Utilizing the systems andmethods described herein, the time and labor required to preparesequencing libraries is reduced in comparison with conventionaltechniques.

It is understood that the invention is not limited to the particularmethodology, protocols, and reagents, etc., described herein, as thesecan be varied by the skilled artisan. It is also understood that theterminology used herein is used for the purpose of describing particularillustrative embodiments only, and is not intended to limit the scope ofthe invention. It also noted that as used herein and in the appendedclaims, the singular forms “a,” “an,” and “the” include the pluralreference unless the context clearly dictates otherwise. Thus, forexample, a reference to “a cell” is a reference to one or more cells andequivalents thereof known to those skilled in the art.

The embodiments of the invention and the various features andadvantageous details thereof are explained more fully with reference tothe non-limiting embodiments and examples that are described and/orillustrated in the accompanying drawings and detailed in the followingdescription. It should be noted that the features illustrated in thedrawings are not necessarily drawn to scale, and features of oneembodiment may be employed with other embodiments as the skilled artisanwould recognize, even if not explicitly stated herein. Descriptions ofwell-known components and processing techniques may be omitted so as tonot unnecessarily obscure the embodiments of the invention.

Definitions

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified. These terms are defined specificallyfor clarity, but all of the definitions are consistent with how askilled artisan would understand these terms.

The term “adjacent,” when used herein to refer two nucleotide sequencesin a nucleic acid, can refer to nucleotide sequences separated by 0 toabout 20 nucleotides, more specifically, in a range of about 1 to about10 nucleotides, or sequences that directly abut one another.

The term “nucleic acid” refers to a nucleotide polymer, and unlessotherwise limited, includes known analogs of natural nucleotides thatcan function in a similar manner (e.g., hybridize) to naturallyoccurring nucleotides.

The term nucleic acid includes any form of DNA or RNA, including, forexample, genomic DNA; complementary DNA (cDNA), which is a DNArepresentation of mRNA, usually obtained by reverse transcription ofmessenger RNA (mRNA) or by amplification; DNA molecules producedsynthetically or by amplification; and mRNA.

The term nucleic acid encompasses double- or triple-stranded nucleicacids, as well as single-stranded molecules. In double- ortriple-stranded nucleic acids, the nucleic acid strands need not becoextensive (i.e, a double-stranded nucleic acid need not bedouble-stranded along the entire length of both strands).

The term nucleic acid also encompasses any chemical modificationthereof, such as by methylation and/or by capping. Nucleic acidmodifications can include addition of chemical groups that incorporateadditional charge, polarizability, hydrogen bonding, electrostaticinteraction, and functionality to the individual nucleic acid bases orto the nucleic acid as a whole. Such modifications may include basemodifications such as 2′-position sugar modifications, 5-positionpyrimidine modifications, 8-position purine modifications, modificationsat cytosine exocyclic amines, substitutions of 5-bromo-uracil, backbonemodifications, unusual base pairing combinations such as the isobasesisocytidine and isoguanidine, and the like.

More particularly, in certain embodiments, nucleic acids, can includepolydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), and any other type of nucleicacid that is an N- or C-glycoside of a purine or pyrimidine base, aswell as other polymers containing nonnucleotidic backbones, for example,polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholino(commercially available from the Anti-Virals, Inc., Corvallis, Oreg., asNeugene) polymers, and other synthetic sequence-specific nucleic acidpolymers providing that the polymers contain nucleobases in aconfiguration which allows for base pairing and base stacking, such asis found in DNA and RNA. The term nucleic acid also encompasses linkednucleic acids (LNAs), which are described in U.S. Pat. Nos. 6,794,499,6,670,461, 6,262,490, and 6,770,748, which are incorporated herein byreference in their entirety for their disclosure of LNAs.

The nucleic acid(s) can be derived from a completely chemical synthesisprocess, such as a solid phase-mediated chemical synthesis, from abiological source, such as through isolation from any species thatproduces nucleic acid, or from processes that involve the manipulationof nucleic acids by molecular biology tools, such as DNA replication,PCR amplification, reverse transcription, or from a combination of thoseprocesses.

The term “target nucleic acids” is used herein to refer to particularnucleic acids to be detected in the methods of the invention.

As used herein the term “target nucleotide sequence” refers to amolecule that includes the nucleotide sequence of a target nucleic acid,such as, for example, the amplification product obtained by amplifying atarget nucleic acid or the cDNA produced upon reverse transcription ofan RNA target nucleic acid.

As used herein, the term “complementary” refers to the capacity forprecise pairing between two nucleotides. I.e., if a nucleotide at agiven position of a nucleic acid is capable of hydrogen bonding with anucleotide of another nucleic acid, then the two nucleic acids areconsidered to be complementary to one another at that position.Complementarity between two single-stranded nucleic acid molecules maybe “partial,” in which only some of the nucleotides bind, or it may becomplete when total complementarity exists between the single-strandedmolecules. The degree of complementarity between nucleic acid strandshas significant effects on the efficiency and strength of hybridizationbetween nucleic acid strands.

“Specific hybridization” refers to the binding of a nucleic acid to atarget nucleotide sequence in the absence of substantial binding toother nucleotide sequences present in the hybridization mixture underdefined stringency conditions. Those of skill in the art recognize thatrelaxing the stringency of the hybridization conditions allows sequencemismatches to be tolerated.

In particular embodiments, hybridizations are carried out understringent hybridization conditions. The phrase “stringent hybridizationconditions” generally refers to a temperature in a range from about 5°C. to about 20° C. or 25° C. below than the melting temperature (T_(m))for a specific sequence at a defined ionic strength and pH. As usedherein, the T_(m) is the temperature at which a population ofdouble-stranded nucleic acid molecules becomes half-dissociated intosingle strands. Methods for calculating the T_(m) of nucleic acids arewell known in the art (see, e.g., Berger and Kimmel (1987) METHODS INENZYMOLOGY, VOL. 152: GUIDE TO MOLECULAR CLONING TECHNIQUES, San Diego:Academic Press, Inc. and Sambrook et al. (1989) MOLECULAR CLONING: ALABORATORY MANUAL, 2ND ED., VOLS. 1-3, Cold Spring Harbor Laboratory),both incorporated herein by reference). As indicated by standardreferences, a simple estimate of the T_(m) value may be calculated bythe equation: T_(m)=81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (see, e.g., Anderson and Young, Quantitative FilterHybridization in NUCLEIC ACID HYBRIDIZATION (1985)). The meltingtemperature of a hybrid (and thus the conditions for stringenthybridization) is affected by various factors such as the length andnature (DNA, RNA, base composition) of the primer or probe and nature ofthe target nucleic acid (DNA, RNA, base composition, present in solutionor immobilized, and the like), as well as the concentration of salts andother components (e.g., the presence or absence of formamide, dextransulfate, polyethylene glycol). The effects of these factors are wellknown and are discussed in standard references in the art. Illustrativestringent conditions suitable for achieving specific hybridization ofmost sequences are: a temperature of at least about 60° C. and a saltconcentration of about 0.2 molar at pH7.

The term “oligonucleotide” is used to refer to a nucleic acid that isrelatively short, generally shorter than 200 nucleotides, moreparticularly, shorter than 100 nucleotides, most particularly, shorterthan 50 nucleotides. Typically, oligonucleotides are single-stranded DNAmolecules.

The term “primer” refers to an oligonucleotide that is capable ofhybridizing (also termed “annealing”) with a nucleic acid and serving asan initiation site for nucleotide (RNA or DNA) polymerization underappropriate conditions (i.e., in the presence of four differentnucleoside triphosphates and an agent for polymerization, such as DNA orRNA polymerase or reverse transcriptase) in an appropriate buffer and ata suitable temperature. The appropriate length of a primer depends onthe intended use of the primer, but primers are typically at least 7nucleotides long and, more typically range from 10 to 30 nucleotides, oreven more typically from 15 to 30 nucleotides, in length. Other primerscan be somewhat longer, e.g., 30 to 50 nucleotides long. In thiscontext, “primer length” refers to the portion of an oligonucleotide ornucleic acid that hybridizes to a complementary “target” sequence andprimes nucleotide synthesis. Short primer molecules generally requirecooler temperatures to form sufficiently stable hybrid complexes withthe template. A primer need not reflect the exact sequence of thetemplate but must be sufficiently complementary to hybridize with atemplate. The term “primer site” or “primer binding site” refers to thesegment of the target nucleic acid to which a primer hybridizes.

A primer is said to anneal to another nucleic acid if the primer, or aportion thereof, hybridizes to a nucleotide sequence within the nucleicacid. The statement that a primer hybridizes to a particular nucleotidesequence is not intended to imply that the primer hybridizes eithercompletely or exclusively to that nucleotide sequence. For example, incertain embodiments, amplification primers used herein are said to“anneal to a nucleotide tag.” This description encompasses primers thatanneal wholly to the nucleotide tag, as well as primers that annealpartially to the nucleotide tag and partially to an adjacent nucleotidesequence, e.g., a target nucleotide sequence. Such hybrid primers canincrease the specificity of the amplification reaction.

As used herein, the selection of primers “so as to avoid substantialannealing to the target nucleic acids” means that primers are selectedso that the majority of the amplicons detected after amplification are“full-length” in the sense that they result from priming at the expectedsites at each end of the target nucleic acid, as opposed to ampliconsresulting from priming within the target nucleic acid, which producesshorter-than-expected amplicons. In various embodiments, primers areselected to that at least 55%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% are full-length.

The term “primer pair” refers to a set of primers including a 5′“upstream primer” or “forward primer” that hybridizes with thecomplement of the 5′ end of the DNA sequence to be amplified and a 3′“downstream primer” or “reverse primer” that hybridizes with the 3′ endof the sequence to be amplified. As will be recognized by those of skillin the art, the terms “upstream” and “downstream” or “forward” and“reverse” are not intended to be limiting, but rather provideillustrative orientation in particular embodiments.

A “probe” is a nucleic acid capable of binding to a target nucleic acidof complementary sequence through one or more types of chemical bonds,generally through complementary base pairing, usually through hydrogenbond formation, thus forming a duplex structure. The probe binds orhybridizes to a “probe binding site.” The probe can be labeled with adetectable label to permit facile detection of the probe, particularlyonce the probe has hybridized to its complementary target.Alternatively, however, the probe may be unlabeled, but may bedetectable by specific binding with a ligand that is labeled, eitherdirectly or indirectly. Probes can vary significantly in size.Generally, probes are at least 7 to 15 nucleotides in length. Otherprobes are at least 20, 30, or 40 nucleotides long. Still other probesare somewhat longer, being at least 50, 60, 70, 80, or 90 nucleotideslong. Yet other probes are longer still, and are at least 100, 150, 200or more nucleotides long. Probes can also be of any length that iswithin any range bounded by any of the above values (e.g., 15-20nucleotides in length).

The primer or probe can be perfectly complementary to the target nucleicacid sequence or can be less than perfectly complementary. In certainembodiments, the primer has at least 65% identity to the complement ofthe target nucleic acid sequence over a sequence of at least 7nucleotides, more typically over a sequence in the range of 10-30nucleotides, and often over a sequence of at least 14-25 nucleotides,and more often has at least 75% identity, at least 85% identity, atleast 90% identity, or at least 95%, 96%, 97%. 98%, or 99% identity. Itwill be understood that certain bases (e.g., the 3′ base of a primer)are generally desirably perfectly complementary to corresponding basesof the target nucleic acid sequence. Primer and probes typically annealto the target sequence under stringent hybridization conditions.

The term “nucleotide tag” is used herein to refer to a predeterminednucleotide sequence that is added to a target nucleotide sequence. Thenucleotide tag can encode an item of information about the targetnucleotide sequence, such the identity of the target nucleotide sequenceor the identity of the sample from which the target nucleotide sequencewas derived. In certain embodiments, such information may be encoded inone or more nucleotide tags, e.g., a combination of two nucleotide tags,one on either end of a target nucleotide sequence, can encode theidentity of the target nucleotide sequence.

As used herein the term “barcode primer” refers to a primer thatincludes a specific barcode nucleotide sequence that encodes informationabout the amplicon produced when the barcode primer is employed in anamplification reaction. For example, a different barcode primer can beemployed to amplify one or more target sequences from each of a numberof different samples, such that the barcode nucleotide sequenceindicates the sample origin of the resulting amplicons.

As used herein, the term “encoding reaction” refers to reaction in whichat least one nucleotide tag is added to a target nucleotide sequence.Nucleotide tags can be added, for example, by an “encoding PCR” in whichthe at least one primer comprises a target-specific portion and anucleotide tag located on the 5′ end of the target-specific portion, anda second primer that comprises only a target-specific portion or atarget-specific portion and a nucleotide tag located on the 5′ end ofthe target-specific portion. For illustrative examples of PCR protocolsapplicable to encoding PCR, see pending WO Application US03/37808 aswell as U.S. Pat. No. 6,605,451. Nucleotide tags can also be added by an“encoding ligation” reaction that can comprise a ligation reaction inwhich at least one primer comprises a target-specific portion andnucleotide tag located on the 5′ end of the target-specific portion, anda second primer that comprises a target-specific portion only or atarget-specific portion and a nucleotide tag located on the 5′ end ofthe target specific portion. Illustrative encoding ligation reactionsare described, for example, in U.S. Patent Publication No. 2005/0260640,which is hereby incorporated by reference in its entirety, and inparticular for ligation reactions.

As used herein an “encoding reaction” produces a “tagged targetnucleotide sequence,” which includes a nucleotide tag linked to a targetnucleotide sequence.

As used herein with reference to a portion of a primer, the term“target-specific” nucleotide sequence refers to a sequence that canspecifically anneal to a target nucleic acid or a target nucleotidesequence under suitable annealing conditions.

As used herein with reference to a portion of a primer, the term“nucleotide tag-specific nucleotide sequence” refers to a sequence thatcan specifically anneal to a nucleotide tag under suitable annealingconditions.

Amplification according to the present teachings encompasses any meansby which at least a part of at least one target nucleic acid isreproduced, typically in a template-dependent manner, including withoutlimitation, a broad range of techniques for amplifying nucleic acidsequences, either linearly or exponentially. Illustrative means forperforming an amplifying step include ligase chain reaction (LCR),ligase detection reaction (LDR), ligation followed by Q-replicaseamplification, PCR, primer extension, strand displacement amplification(SDA), hyperbranched strand displacement amplification, multipledisplacement amplification (MDA), nucleic acid strand-basedamplification (NASBA), two-step multiplexed amplifications, rollingcircle amplification (RCA), and the like, including multiplex versionsand combinations thereof, for example but not limited to, OLA/PCR,PCR/OLA, LDR/PCR, PCR/PCR/LDR, PCR/LDR, LCR/PCR, PCR/LCR (also known ascombined chain reaction—CCR), and the like. Descriptions of suchtechniques can be found in, among other sources, Ausbel et al.; PCRPrimer: A Laboratory Manual, Diffenbach, Ed., Cold Spring Harbor Press(1995); The Electronic Protocol Book, Chang Bioscience (2002); Msuih etal., J. Clin. Micro. 34:501-07 (1996); The Nucleic Acid ProtocolsHandbook, R. Rapley, ed., Humana Press, Totowa, N.J. (2002); Abramson etal., Curr Opin Biotechnol. 1993 February; 4(1):41-7, U.S. Pat. No.6,027,998; U.S. Pat. No. 6,605,451, Barany et al., PCT Publication No.WO 97/31256; Wenz et al., PCT Publication No. WO 01/92579; Day et al.,Genomics, 29(1): 152-162 (1995), Ehrlich et al., Science 252:1643-50(1991); Innis et al., PCR Protocols: A Guide to Methods andApplications, Academic Press (1990); Favis et al., Nature Biotechnology18:561-64 (2000); and Rabenau et al., Infection 28:97-102 (2000);Belgrader, Barany, and Lubin, Development of a Multiplex LigationDetection Reaction DNA Typing Assay, Sixth International Symposium onHuman Identification, 1995 (available on the world wide web at:promega.com/geneticidproc/ussymp6proc/blegrad.html-); LCR KitInstruction Manual, Cat. #200520, Rev. #050002, Stratagene, 2002;Barany, Proc. Natl. Acad. Sci. USA 88:188-93 (1991); Bi and Sambrook,Nucl. Acids Res. 25:2924-2951 (1997); Zirvi et al., Nucl. Acid Res.27:e40i-viii (1999); Dean et al., Proc Natl Acad Sci USA 99:5261-66(2002); Barany and Gelfand, Gene 109:1-11 (1991); Walker et al., Nucl.Acid Res. 20:1691-96 (1992); Polstra et al., BMC Inf. Dis. 2:18- (2002);Lage et al., Genome Res. 2003 February; 13(2):294-307, and Landegren etal., Science 241:1077-80 (1988), Demidov, V., Expert Rev Mol Diagn. 2002November; 2(6):542-8., Cook et al., J Microbiol Methods. 2003 May;53(2):165-74, Schweitzer et al., Curr Opin Biotechnol. 2001 February;12(1):21-7, U.S. Pat. No. 5,830,711, U.S. Pat. No. 6,027,889, U.S. Pat.No. 5,686,243, PCT Publication No. WO0056927A3, and PCT Publication No.WO9803673A1.

In some embodiments, amplification comprises at least one cycle of thesequential procedures of: annealing at least one primer withcomplementary or substantially complementary sequences in at least onetarget nucleic acid; synthesizing at least one strand of nucleotides ina template-dependent manner using a polymerase; and denaturing thenewly-formed nucleic acid duplex to separate the strands. The cycle mayor may not be repeated. Amplification can comprise thermocycling or canbe performed isothermally.

The term “qPCR” is used herein to refer to quantitative real-timepolymerase chain reaction (PCR), which is also known as “real-time PCR”or “kinetic polymerase chain reaction.”

A “reagent” refers broadly to any agent used in a reaction, other thanthe analyte (e.g., nucleic acid being analyzed). Illustrative reagentsfor a nucleic acid amplification reaction include, but are not limitedto, buffer, metal ions, polymerase, reverse transcriptase, primers,template nucleic acid, nucleotides, labels, dyes, nucleases, and thelike. Reagents for enzyme reactions include, for example, substrates,cofactors, buffer, metal ions, inhibitors, and activators.

The term “universal detection probe” is used herein to refer to anyprobe that identifies the presence of an amplification product,regardless of the identity of the target nucleotide sequence present inthe product.

The term “universal qPCR probe” is used herein to refer to any suchprobe that identifies the presence of an amplification product duringqPCR. In particular embodiments, nucleotide tags according to theinvention can comprise a nucleotide sequence to which a detection probe,such as a universal qPCR probe binds. Where a tag is added to both endsof a target nucleotide sequence, each tag can, if desired, include asequence recognized by a detection probe. The combination of suchsequences can encode information about the identity or sample source ofthe tagged target nucleotide sequence. In other embodiments, one or moreamplification primers can comprise a nucleotide sequence to which adetection probe, such as a universal qPCR probe binds. In this manner,one, two, or more probe binding sites can be added to an amplificationproduct during the amplification step of the methods of the invention.Those of skill in the art recognize that the possibility of introducingmultiple probe binding sites during preamplification (if carried out)and amplification facilitates multiplex detection, wherein two or moredifferent amplification products can be detected in a givenamplification mixture or aliquot thereof

The term “universal detection probe” is also intended to encompassprimers labeled with a detectable label (e.g., a fluorescent label), aswell as non-sequence-specific probes, such as DNA binding dyes,including double-stranded DNA (dsDNA) dyes, such as SYBR Green.

The term “target-specific qPCR probe” is used herein to refer to a qPCRprobe that identifies the presence of an amplification product duringqPCR, based on hybridization of the qPCR probe to a target nucleotidesequence present in the product.

“Hydrolysis probes” are generally described in U.S. Pat. No. 5,210,015,which is incorporated herein by reference in its entirety for itsdescription of hydrolysis probes. Hydrolysis probes take advantage ofthe 5′-nuclease activity present in the thermostable Taq polymeraseenzyme typically used in the PCR reaction (TaqMan® probe technology,Applied Biosystems, Foster City Calif.). The hydrolysis probe is labeledwith a fluorescent detector dye such as fluorescin, and an acceptor dyeor quencher. In general, the fluorescent dye is covalently attached tothe 5′ end of the probe and the quencher is attached to the 3′ end ofthe probe, and when the probe is intact, the fluorescence of thedetector dye is quenched by fluorescence resonance energy transfer(FRET). The probe anneals downstream of one of the primers that definesone end of the target nucleic acid in a PCR reaction. Using thepolymerase activity of the Taq enzyme, amplification of the targetnucleic acid is directed by one primer that is upstream of the probe anda second primer that is downstream of the probe but anneals to theopposite strand of the target nucleic acid. As the upstream primer isextended, the Taq polymerase reaches the region where the labeled probeis annealed, recognizes the probe-template hybrid as a substrate, andhydrolyzes phosphodiester bonds of the probe. The hydrolysis reactionirrevocably releases the quenching effect of the quencher dye on thereporter dye, thus resulting in increasing detector fluorescence witheach successive PCR cycle. In particular, hydrolysis probes suitable foruse in the invention can be capable of detecting 8-mer or 9-mer motifsthat are common in the human and other genomes and/or transcriptomes andcan have a high T_(m) of about 70° C. enabled by the use of linkednucleic acid (LNA) analogs.

The term “label,” as used herein, refers to any atom or molecule thatcan be used to provide a detectable and/or quantifiable signal. Inparticular, the label can be attached, directly or indirectly, to anucleic acid or protein. Suitable labels that can be attached to probesinclude, but are not limited to, radioisotopes, fluorophores,chromophores, mass labels, electron dense particles, magnetic particles,spin labels, molecules that emit chemiluminescence, electrochemicallyactive molecules, enzymes, cofactors, and enzyme substrates.

The term “dye,” as used herein, generally refers to any organic orinorganic molecule that absorbs electromagnetic radiation at awavelength greater than or equal 340 nm.

The term “fluorescent dye,” as used herein, generally refers to any dyethat emits electromagnetic radiation of longer wavelength by afluorescent mechanism upon irradiation by a source of electromagneticradiation, such as a lamp, a photodiode, or a laser.

The term “elastomer” has the general meaning used in the art. Thus, forexample, Allcock et al. (Contemporary Polymer Chemistry, 2nd Ed.)describes elastomers in general as polymers existing at a temperaturebetween their glass transition temperature and liquefaction temperature.Elastomeric materials exhibit elastic properties because the polymerchains readily undergo torsional motion to permit uncoiling of thebackbone chains in response to a force, with the backbone chainsrecoiling to assume the prior shape in the absence of the force. Ingeneral, elastomers deform when force is applied, but then return totheir original shape when the force is removed.

A “polymorphic marker” or “polymorphic site” is a locus at whichnucleotide sequence divergence occurs. Illustrative markers have atleast two alleles, each occurring at frequency of greater than 1%, andmore typically greater than 10% or 20% of a selected population. Apolymorphic site may be as small as one base pair. Polymorphic markersinclude restriction fragment length polymorphism (RFLPs), variablenumber of tandem repeats (VNTR's), hypervariable regions,minisatellites, dinucleotide repeats, trinucleotide repeats,tetranucleotide repeats, simple sequence repeats, deletions, andinsertion elements such as Alu. The first identified allelic form isarbitrarily designated as the reference form and other allelic forms aredesignated as alternative or variant alleles. The allelic form occurringmost frequently in a selected population is sometimes referred to as thewildtype form. Diploid organisms may be homozygous or heterozygous forallelic forms. A diallelic polymorphism has two forms. A triallelicpolymorphism has three forms.

A “single nucleotide polymorphism” (SNP) occurs at a polymorphic siteoccupied by a single nucleotide, which is the site of variation betweenallelic sequences. The site is usually preceded by and followed byhighly conserved sequences of the allele (e.g., sequences that vary inless than 1/100 or 1/1000 members of the populations). A SNP usuallyarises due to substitution of one nucleotide for another at thepolymorphic site. A transition is the replacement of one purine byanother purine or one pyrimidine by another pyrimidine. A transversionis the replacement of a purine by a pyrimidine or vice versa. SNPs canalso arise from a deletion of a nucleotide or an insertion of anucleotide relative to a reference allele.

Amplification Methods

In General

In particular embodiments, the invention provides an amplificationmethod for introducing a plurality (e.g., at least three) of selectednucleotide sequences into one or more target nucleic acid(s). The methodentails amplifying a plurality of target nucleic acids, typically, in aplurality of samples. In illustrative embodiments, the same set oftarget nucleic acids can be amplified in each of two or more differentsamples. The samples can differ from one another in any way, e.g., thesamples can be from different tissues, subjects, environmental sources,etc. At least three primers can be used to amplify each target nucleicacid, namely: forward and reverse amplification primers, each primerincluding a target-specific portion and one or both primers including anucleotide tag. The target-specific portions can specifically anneal toa target under suitable annealing conditions. The nucleotide tag for theforward primer can have a sequence that is the same as, or differentfrom, the nucleotide tag for the reverse primer. Generally, thenucleotide tags are 5′ of the target-specific portions. The third primeris a barcode primer comprising a barcode nucleotide sequence and a firstand/or second nucleotide tag-specific portion. The barcode nucleotidesequence is a sequence selected to encode information about the ampliconproduced when the barcode primer is employed in an amplificationreaction. The tag-specific portion can specifically anneal to the one orboth nucleotide tags in the forward and reverse primers. The barcodeprimer is generally 5′ of the tag-specific portion.

The barcode primer is typically present in the amplification mixture inexcess of the forward and/or reverse primer(s). More specifically, ifthe barcode primer anneals to the nucleotide tag in the forward primer,the barcode primer is generally present in excess of the forward primer.If the barcode primer anneals to the nucleotide tag in the reverseprimer, the barcode primer is generally present in excess of the reverseprimer. In each instance the third primer in the amplification mixture,i.e., the reverse primer or the forward primer, respectively, can bepresent, in illustrative embodiments, at a concentration approximatelysimilar to that of the barcode primer. Generally the barcode primer ispresent in substantial excess. For example, the concentration of thebarcode primer in the amplification mixtures can be at least 2-fold, atleast 4-fold, at least 5-fold, at least 10-fold, at least 15-fold, atleast 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, atleast 40-fold, at least 45-fold, at least 50-fold, at least 100-fold, atleast 500-fold, at least 10³-fold, at least 5×10³-fold, at least10⁴-fold, at least 5×10⁴-fold, at least 10⁵-fold, at least 5×10⁵-fold,at least 10⁶-fold, or higher, relative to the concentration of theforward and/or reverse primer(s). In addition, the concentration excessof the barcode primer can fall within any range having any of the abovevalues as endpoints (e.g., 2-fold to 10⁵-fold). In illustrativeembodiments, where the barcode primer has a tag-specific portion that isspecific for the nucleotide tag on the forward primer, the forwardprimer can be present in picomolar to nanomolar concentrations, e.g.,about 5 pM to 500 nM, about 5 pM to 100 nM, about 5 pM to 50 nM, about 5pM to 10 nM, about 5 pM to 5 nM, about 10 pM to 1 nM, about 50 pM toabout 500 pM, about 100 pM or any other range having any of these valuesas endpoints (e.g., 10 pM to 50 pM). Suitable, illustrativeconcentrations of barcode primer that could be used on combination withany of these concentrations of forward primer include about 10 nM toabout 10 μM, about 25 nM to about 7.5 μM, about 50 nM to about 5 μM,about 75 nM to about 2.5 μM, about 100 nM to about 1 μM, about 250 nM toabout 750 nM, about 500 nM or any other range having any of these valuesas endpoints (e.g., 100 nM to 500 nM). In amplification reactions usingsuch concentrations of forward and barcode primers, the reverse primerhave a concentration on the same order as the barcode primer (e.g.within about 10-fold, within about 5-fold, or equal).

Each amplification mixture can be subjected to amplification to producetarget amplicons comprising tagged target nucleotide sequences, eachcomprising first and second nucleotide tags flanking the targetnucleotide sequence, and at least one barcode nucleotide sequence at the5′ or 3′ end of the target amplicon (relative to one strand of thetarget amplicon). In certain embodiments, the first and secondnucleotide tags and/or the barcode nucleotide sequence are selected soas to avoid substantial annealing to the target nucleic acids. In suchembodiments, the tagged target nucleotide sequences can includemolecules having the following elements: 5′-(barcode nucleotidesequence)-(first nucleotide tag from the forward primer)-(targetnucleotide sequence)-(second nucleotide tag sequence from the reverseprimer)-3′ or 5′-(first nucleotide tag from the forward primer)-(targetnucleotide sequence)-(second nucleotide tag sequence from the reverseprimer)-(barcode nucleotide sequence)-3′.

In illustrative embodiments, the barcode nucleotide sequence identifiesa particular sample. Thus, for example, a set of T target nucleic acidscan be amplified in each of S samples, where S and T are integers,typically greater than one. In such embodiments, amplification can beperformed separately for each sample, wherein the same set of forwardand reverse primers is used for each sample and the set of forward andreverse primers has at least one nucleotide tag that is common to allprimers in the set. A different barcode primer can be used for eachsample, wherein the bar code primers have different barcode nucleotidesequences, but the same tag-specific portion that can anneal to thecommon nucleotide tag. This embodiment has the advantage of reducing thenumber of different primers that would need to be synthesized to encodesample origin in amplicons produced for a plurality of target sequences.Alternatively, different sets of forward and reverse primers can beemployed for each sample, wherein each set has a nucleotide tag that isdifferent from the primers in the other set, and different barcodeprimers are used for each sample, wherein the barcode primers havedifferent barcode nucleotide sequences and different tag-specificportions. In either case, the amplification produces a set of Tamplicons from each sample that bear sample-specific barcodes.

In embodiments, wherein the same set of forward and reverse primers isused for each sample, the forward and reverse primers for each targetcan be initially combined separately from the sample, and each barcodeprimer can be initially combined with its corresponding sample. Aliquotsof the initially combined forward and reverse primers can then be addedto aliquots of the initially combined sample and barcode primer toproduce S×T amplification mixtures. These amplification mixtures can beformed in any article that can be subjected to conditions suitable foramplification. For example, the amplification mixtures can be formed in,or distributed into, separate compartments of a microfluidic deviceprior to amplification. Suitable microfluidic devices include, inillustrative embodiments, matrix-type microfluidic devices, such asthose described below.

Any amplification method can be employed to produce amplicons from theamplification mixtures. In illustrative embodiments, PCR is employed.The amplification is generally carried out for at least three cycles tointroduce the first and second nucleotide tags and the barcodenucleotide sequence. In various embodiments, amplification is carriedout for 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 cycles, or for anynumber of cycles falling within a range having any of these values asendpoints (e.g. 5-10 cycles). In particular embodiments, amplificationis carried out for a sufficient number of cycles to normalize targetamplicon copy number across targets and across samples (e.g., 15, 20,25, 30, 35, 40, 45, or 50 cycles, or for any number of cycles fallingwithin a range having any of these values as endpoints).

Particular embodiments of the above-described method providesubstantially uniform amplification, yielding a plurality of targetamplicons wherein the majority of amplicons are present at a levelrelatively close to the average copy number calculated for the pluralityof target amplicons. Thus, in various embodiments, at least 50, at least55, at least 60, at least 65, at least 70, at least 75, at least 80, atleast 85, at least 90, at least 91, at least 92, at least 93, at least94, at least 95, at least 96, at least 97, at least 98, or at least 99percent of the target amplicons are present at greater than 50 percentof the average number of copies of target amplicons and less than 2-foldthe average number of copies of target amplicons.

The invention also provides, in certain embodiments, a method foramplifying a plurality of target nucleotides in which barcoding is,optionally, omitted and the target nucleotide sequences are tagged afteramplification. More specifically, the invention provides a method foramplifying a plurality of target nucleic acids, typically, in aplurality of samples, that entails preparing an amplification mixturefor each target nucleic acid. Each amplification mixture includes aforward primer including a target-specific sequence and a reverse primerincluding a target-specific sequence. The amplification mixtures aresubjected to amplification to produce a plurality of target nucleotidesequences. The target nucleotide sequences are then tagged to produce aplurality of target amplicons, each including first and/or secondnucleotide tags flanking the target nucleotide sequence. This methodproduces a plurality of target amplicons, wherein at least 50 percent ofthe target amplicons are present at greater than 50 percent of theaverage number of copies of target amplicons and less than 2-fold theaverage number of copies of target amplicons. In various embodiments ofthis method at least 55, at least 60, at least 65, at least 70, at least75, at least 80, at least 85, at least 90, at least 91, at least 92, atleast 93, at least 94, at least 95, at least 96, at least 97, at least98, or at least 99 percent of the target amplicons are present atgreater than 50 percent of the average number of copies of targetamplicons and less than 2-fold the average number of copies of targetamplicons.

In various embodiments, the target nucleotide sequence amplified can be,e.g., 25 bases, 50 bases, 100 bases, 200 bases, 500 bases, or 750 bases.In certain embodiments of the above-described methods, a long-rangeamplification method, such as long-range PCR can be employed to produceamplicons from the amplification mixtures. Long-range PCR permits theamplification of target nucleotide sequences ranging from one or a fewkilobases (kb) to over 50 kb. In various embodiments, the targetnucleotide sequences that are amplified by long-range PCR are at leastabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35,40, 45, or 50 kb in length. Target nucleotide sequences can also fallwithin any range having any of these values as endpoints (e.g., 25 basesto 100 bases or 5-15 kb). The use of long-range PCR in theabove-described methods can, in some embodiments, yield a plurality oftarget amplicons wherein at least 50, at least 55, at least 60, at least65, or at least 70 percent of the target amplicons are present atgreater than 50 percent of the average number of copies of targetamplicons and less than 2-fold the average number of copies of targetamplicons.

Long-range PCR is well known in the art. See, e.g., Cheng S, Fockler C,Barnes W M, Higuchi R (June 1994). “Effective amplification of longtargets from cloned inserts and human genomic DNA”. Proc. Natl. Acad.Sci. U.S.A. 91 (12): 5695-9. Enzymes, protocols, and kits for long-rangePCR that are suitable for use in the methods described here arecommercially available; examples include: SequalPrep™ Long PCR Kit(Invitrogen, USA), PfuUltra® II Fusion HS DNA polymerase (Stratagene),Phusion® DNA polymerases, Phusion® Flash High Fidelity PCR Master Mix(Finnzymes).

In certain embodiments, the target amplicons can be recovered from theamplification mixtures. For example, a matrix-type microfluidic devicethat is adapted to permit recovery of the contents of each reactionchamber (see below) can be employed for the amplification to generatethe target amplicons. In variations of these embodiments, the targetamplicons can be subjected to further amplification and/or analysis. Forexample, one or more target amplicon(s) can be subjected toamplification using primers specific for the first and second nucleotidetags to produce a target amplicon lacking the barcode nucleotidesequence. In certain embodiments, the amount of target ampliconsproduced in the amplification mixtures can be quantified duringamplication, e.g., by quantitative real-time PCR, or after.

In particular embodiments, the above-described amplification methods areemployed to produce amplicons suitable for automated DNA sequencing. Inparticular, the ability of the methods to provide substantially uniformamplification, as described above, of target nucleotide sequences ishelpful in preparing DNA sequencing libraries having good coverage. Inthe context of automated DNA sequencing, the term “coverage” refers tothe number of times the sequence is measured upon sequencing. A DNAsequencing library that has substantially uniform coverage can yieldsequence data where the coverage is also substantially uniform. Thus, invarious embodiments, upon performing automated sequencing of a pluralityof target amplicons prepared as described herein, the sequences of atleast 50 percent of the target amplicons are present at greater than 50percent of the average number of copies of target amplicon sequences andless than 2-fold the average number of copies of target ampliconsequences. In various embodiments of this method at least 55, at least60, at least 65, at least 70, at least 75, at least 80, at least 85, atleast 90, at least 91, at least 92, at least 93, at least 94, at least95, at least 96, at least 97, at least 98, or at least 99 percent of thetarget amplicon sequences are present at greater than 50 percent of theaverage number of copies of target amplicon sequences and less than2-fold the average number of copies of target amplicon sequences.

Preparation of Nucleic Acids for DNA Sequencing

Many current DNA sequencing techniques rely on “sequencing bysynthesis.” These techniques entail library creation, massively parallelPCR amplification of library molecules, and sequencing. Library creationstarts with conversion of sample nucleic acids to appropriately sizedfragments, ligation of adaptor sequences onto the ends of the fragments,and selection for molecules properly appended with adaptors. Thepresence of the adaptor sequences on the ends of the library moleculesenables amplification of random-sequence inserts. The above-describedmethods for tagging nucleotide sequences can be substituted forligation, to introduce adaptor sequences, as described in greater detailbelow.

In particular embodiments, the number of library DNA molecules producedin the massively parallel PCR step is low enough that the chance of twomolecules associating with the same substrate, e.g. the same bead (in454 DNA sequencing) or the same surface patch (in Solexa DNA sequencing)is low, but high enough so that the yield of amplified sequences issufficient to provide a high throughput. As discussed further below,after suitable adaptor sequences are introduced, digital PCR can beemployed to calibrate the number of library DNA molecules prior tosequencing by synthesis.

Addition of DNA Sequencing Primers to Nucleic Acids

The DNA to be sequenced can be any type of DNA. In particularembodiments, the DNA is genomic DNA from an organism. In variations ofsuch embodiments, total genomic DNA obtained from a sample taken from anorganism or from a DNA library is prepared for sequencing.

As described above, at least three primers are employed to prepare theDNA for sequencing: forward, reverse, and barcode primers. However, oneor more of the forward primer, reverse primer, and barcode primerincludes at least one additional primer binding site. In specificembodiments, the barcode primer includes at least a first additionalprimer binding site upstream of the barcode nucleotide sequence, whichis upstream of the first nucleotide tag-specific portion. In certainembodiments, two of the forward primer, reverse primer, and barcodeprimer include at least one additional primer binding site (i.e, suchthat the amplicon produced upon amplification includes the nucleotidetag sequences, the barcode nucleotide sequence, and the two additionalbinding sites). For example, if the barcode primer includes a firstadditional primer binding site upstream of the barcode nucleotidesequence, in specific embodiments, the reverse primer can include atleast a second additional primer binding site downstream of the secondnucleotide tag. Amplification then yields a molecule having thefollowing elements: 5′-(first additional primer binding site)-(barcodenucleotide sequence)-(first nucleotide tag from the forwardprimer)-(target nucleotide sequence)-(second nucleotide tag from thereverse primer)-(second additional primer binding site)-3′. In specificembodiments, the first and second additional primer binding sites arecapable of being bound by DNA sequencing primers, to facilitatesequencing of the entire amplicon, including the barcode, which can, asdiscussed above, indicate sample origin.

In some embodiments, more than three primers can be employed to adddesired elements to a target nucleotide sequence. For example, fourprimers can be employed to produce molecules having the same fiveelements discussed above, plus an optional additional barcode e.g.,5′-(first additional primer binding site)-(barcode nucleotidesequence)-(first nucleotide tag from the forward primer)-(targetnucleotide sequence)-(second nucleotide tag from the reverseprimer)-(additional barcode nucleotide sequence)-(second additionalprimer binding site)-3′. In an illustrative four-primer embodiment, theforward primer includes a target-specific portion and first nucleotidetag, and the reverse primer includes a target-specific portion and asecond nucleotide tag. Together, these two primers constitute the “innerprimers.” The remaining two primers are the “outer primers,” whichanneal to the first and second nucleotide tags present in the innerprimers. One outer primer is the barcode primer, which can contain atleast a first additional primer binding site upstream of the barcodenucleotide sequence, which is upstream of the first nucleotidetag-specific portion (i.e., the same barcode primer discussed in theprevious paragraph). The second outer primer can include a secondtag-specific portion, an additional barcode nucleotide sequence and,downstream of this, a second additional primer binding site.

Amplification to incorporate elements from more than three primers canbe carried out in one or multiple amplification reactions. For example,a four-primer amplification can be carried out in one amplificationreaction, in which all four primers are present. Alternatively, afour-primer amplification can be carried out, e.g., in two amplificationreactions: one to incorporate the inner primers and a separateamplification reaction to incorporate the outer primers. Where all fourprimers are present in one amplification reaction, the outer primers aregenerally present in the reaction mixture in excess. The relativeconcentration values give above for the barcode primer relative to theforward and/or reverse primers also apply to the relative concentrationsof the outer primers relative to inner primers in a one-step,four-primer amplification reaction.

In an illustrative embodiment of the four-primer amplification reaction,each of the outer primers contains a unique barcode. For example, onebarcode primer would be constructed of the elements 5′-(first additionalprimer binding site)-(first barcode nucleotide sequence)-(firstnucleotide tag)-3′, and the second barcode primer would be constructedof the elements 5′-(second additional primer binding site)-(secondbarcode nucleotide sequence)-(second nucleotide tag)-3′. In thisembodiment, a number (J) of first barcode primers can be combined with anumber (K) of second barcode primers to create J×K unique amplificationproducts.

In a further illustrative embodiment of the invention, more than fourprimers can be combined in a single reaction to append differentcombinations of additional primer binding sites, barcode sequences, andnucleotide tags. For example, outer barcode primers containing thefollowing elements: 5′-(first additional primer binding site)-(firstbarcode nucleotide sequence)-(first nucleotide tag)-3′, 5′-(firstadditional primer binding site)-(first barcode nucleotidesequence)-(second nucleotide tag)-3′, 5′-(second additional primerbinding site)-(first barcode nucleotide sequence)-(first nucleotidetag)-3′, 5′-(second additional primer binding site)-(first barcodenucleotide sequence)-(second nucleotide tag)-3′, can be combined withinner target-specific primers as described above to produceamplification product pools containing all combinations of the barcodeprimers with the desired amplicon sequence.

In other illustrative embodiments of the invention, outer barcodeprimers in any of the combinations described above, or othercombinations that would be obvious to one of skill in the art, can becombined with more than one pair of target primer sequences bearing thesame first and second nucleotide tag sequences. For example, innerprimers containing up to ten different target-specific forward primersequences combined with the same first nucleotide tag and up to tendifferent target-specific reverse primer sequences combined with thesame second nucleotide tag can be combined with the up to 2 or up to 4outer barcode primers to generate multiple amplification products asdescribed above. In various embodiments, at least 10, at least 20, atleast 50, at least 100, at least 200, at least 500, at least 1000, atleast 2000, at least 5000 or at least 10000 different target-specificprimer pairs bearing the same first nucleotide tag and second nucleotidetag would be combined with the up to 2 or up to 4 outer barcode primersto generate multiple amplification products.

The methods of the invention can include subjecting at least one targetamplicon to DNA sequencing using any available DNA sequencing method. Inparticular embodiments, a plurality of target amplicons is sequencedusing a high throughput sequencing method. Such methods typically use anin vitro cloning step to amplify individual DNA molecules. Emulsion PCR(emPCR) isolates individual DNA molecules along with primer-coated beadsin aqueous droplets within an oil phase. PCR produces copies of the DNAmolecule, which bind to primers on the bead, followed by immobilizationfor later sequencing. emPCR is used in the methods by Marguilis et al.(commercialized by 454 Life Sciences, Branford, Conn.), Shendure andPorreca et al. (also known as “polony sequencing”) and SOLiD sequencing,(Applied Biosystems Inc., Foster City, Calif.). See M. Margulies, et al.(2005) “Genome sequencing in microfabricated high-density picolitrereactors” Nature 437: 376-380; J. Shendure, et al. (2005) “AccurateMultiplex Polony Sequencing of an Evolved Bacterial Genome” Science 309(5741): 1728-1732. In vitro clonal amplification can also be carried outby “bridge PCR,” where fragments are amplified upon primers attached toa solid surface. Braslaysky et al. developed a single-molecule method(commercialized by Helicos Biosciences Corp., Cambridge, Mass.) thatomits this amplification step, directly fixing DNA molecules to asurface. I. Braslaysky, et al. (2003) “Sequence information can beobtained from single DNA molecules” Proceedings of the National Academyof Sciences of the United States of America 100: 3960-3964.

DNA molecules that are physically bound to a surface can be sequenced inparallel. “Sequencing by synthesis,” like dye-terminationelectrophoretic sequencing, uses a DNA polymerase to determine the basesequence. Reversible terminator methods (commercialized by Illumina,Inc., San Diego, Calif. and Helicos Biosciences Corp., Cambridge, Mass.)use reversible versions of dye-terminators, adding one nucleotide at atime, and detect fluorescence at each position in real time, by repeatedremoval of the blocking group to allow polymerization of anothernucleotide. “Pyrosequencing” also uses DNA polymerization, adding onenucleotide at a time and detecting and quantifying the number ofnucleotides added to a given location through the light emitted by therelease of attached pyrophosphates (commercialized by 454 Life Sciences,Branford, Conn.). See M. Ronaghi, et al. (1996). “Real-time DNAsequencing using detection of pyrophosphate release” AnalyticalBiochemistry 242: 84-89.

Sample Preparation by Digital PCR

In some embodiments, samples are loaded into an amplification device,for example, a PCR plate or a microfluidic device, at sampleconcentrations containing on average less than one amplificationtemplate per well or chamber. Each well or chamber in the device isprepared such that it contains suitable tagged target-specific primersand a unique combination of forward and reverse barcode primers. Forexample, one well can contain barcode primers containing the elements5′-(first additional primer binding site)-(first barcodesequence)-(first nucleotide tag)-3′, 5′-(second additional primerbinding site)-(second barcode sequence)-(second nucleotide tag)-3′. Asecond well or chamber can contain barcode primers containing theelements 5′-(first additional primer binding site)-(third barcodesequence)-(first nucleotide tag)-3′, 5′-(second additional primerbinding site)-(fourth barcode sequence)-(second nucleotide tag)-3′.Amplification products produced in each well would be labeled uniquelywith the combinations of barcode sequences loaded into these wells.

Sample Calibration by Digital PCR

In particular embodiments, the number of target amplicons produced, e.g.from a DNA library, using the above-described methods can be calibratedusing a digital amplification method. The step is finds particularapplication in preparing DNA for sequencing by synthesis. Fordiscussions of “digital PCR” see, for example, Vogelstein and Kinzler,1999, Proc Natl Acad Sci USA 96:9236-41; McBride et al., U.S PatentApplication Publication No. 20050252773, especially Example 5 (each ofthese publications are hereby incorporated by reference in theirentirety, and in particular for their disclosures of digitalamplification). Digital amplification methods can make use ofcertain-high-throughput devices suitable for digital PCR, such asmicrofluidic devices typically including a large number and/or highdensity of small-volume reaction sites (e.g., nano-volume reaction sitesor reaction chambers). In illustrative embodiments, digitalamplification is performed using a microfluidic device, such as theDigital Array microfluidic devices described below. Digitalamplification can entail distributing or partitioning a sample amonghundreds to thousands of reaction mixtures disposed in a reaction/assayplatform or microfluidic device. In such embodiments, a limitingdilution of the sample is made across a large number of separateamplification reactions such that most of the reactions have no templatemolecules and give a negative amplification result. In counting thenumber of positive amplification results, e.g, at the reaction endpoint,one is counting the individual template molecules present in the inputsample one-by-one. A major advantage of digital amplification is thatthe quantification is independent of variations in the amplificationefficiency—successful amplifications are counted as one molecule,independent of the actual amount of product.

In certain embodiments, digital amplification can be carried out afterpreamplification of sample nucleic acids. Typically, preamplificationprior to digital amplification is performed for a limited number ofthermal cycles (e.g., 5 cycles, or 10 cycles). In certain embodiments,the number of thermal cycles during preamplification can range fromabout 4 to 15 thermal cycles, or about 4-10 thermal cycles. In certainembodiments the number of thermal cycles can be 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, or more than 15. The above-described amplificationto produce adaptor sequence-containing amplicons for DNA sequencing canbe substituted for the typical preamplification step.

Digital amplication methods are described in U.S. Publication No.20090239308, published Sep. 24, 2009, which is hereby incorporated byreference in its entirety and, in particular, for its disclosure ofdigital amplification methods and devices. Generally, in digitalamplification, identical (or substantially similar) amplificationreactions are run on a nucleic acid sample, such as genomic DNA. Thenumber of individual reactions for a given nucleic acid sample may varyfrom about 2 to over 1,000,000. Typically, the number of reactionsperformed on a sample is about 100 or greater, more typically about 200or greater, and even more typically about 300 or greater. Larger scaledigital amplification can also be performed in which the number ofreactions performed on a sample is about 500 or greater, about 700 orgreater, about 765 or greater, about 1,000 or greater, about 2,500 orgreater, about 5,000 or greater, about 7,500 or greater, or about 10,000or greater. The number of reactions performed may also be significantlyhigher, such up to about 25,000, up to about 50,000, up to about 75,000,up to about 100,000, up to about 250,000, up to about 500,000, up toabout 750,000, up to about 1,000,000, or even greater than 1,000,000assays per genomic sample.

In particular embodiments, the quantity of nucleic acid subjected todigital amplification is generally selected such that, when distributedinto discrete reaction mixtures, each individual amplification reactionis expected to include one or fewer amplifiable nucleic acids. One ofskill in the art can determine the concentration of target amplicon(s)produced as described above and calculate an appropriate amount for usein digital amplification. More conveniently, a set of serial dilutionsof the target amplicon(s) can be tested. For example, a device that iscommercially available from Fluidigm Corp. as the 12.765 Digital Arraymicrofluidic device allows 12 different dilutions to be testedsimultaneously. Optionally, a suitable dilution can be determined bygenerating a linear regression plot. For the optimal dilution, the lineshould be straight and pass through the origin. Subsequently theconcentration of the original samples can be calculated from the plot.

The appropriate quantity of target amplicon(s) can be distributed intodiscrete locations or reaction wells or chambers such that each reactionincludes, for example, an average of no more than about one amplicon pervolume. The target amplicon(s) can be combined with reagents selectedfor quantitative or nonquantitative amplification, prior to distributionor after.

Following distribution, the reaction mixtures are subjected toamplification to identify those reaction mixtures that contained atarget amplicon. Any amplification method can be employed, butconveniently, PCR is used, e.g., real-time PCR or endpoint PCR. Thisamplification can employ any primers capable of amplifying the targetamplicon(s). Thus, in particular embodiments, the primers can be DNAsequencing primers that anneal to the primer binding sites introduced inthe previous amplification step.

The concentration of any target amplicon (copies/μL) is correlated withthe number of positive (i.e., amplification product-containing) reactionmixtures. See copending U.S. application Ser. No. 12/170,414, entitled“Method and Apparatus for Determining Copy Number Variation UsingDigital PCR,” which is incorporated by reference for all purposes, and,in particular, for analysis of digital PCR results. Also see Dube etal., 2008, “Mathematical Analysis of Copy Number Variation in a DNASample Using Digital PCR on a Nanofluidic Device” PLoS ONE 3(8): e2876.doi:10.1371/journal.pone.0002876, which is incorporated by reference forall purposes and, in particular, for analysis of digital PCR results.

In an illustrative embodiment of sample calibration for DNA sequencingby digital PCR, a PCR reaction mix containing roughly 100-360 ampliconsper μl can be loaded onto a Digital Array microfluidic device, such asFluidigm Corporation's (South San Francisco, Calif.) 12.765 DigitalArray microfluidic device, described below. The microfluidic chip has 12panels and each panel contains 765 chambers. Replicate panels on thedigital chip can be assayed in order to obtain absolute quantificationof the initial concentration of library. The diluted samples havingtypical relative coefficients of variation (between replicates) within9-12% (or lower) can be used for sequencing. See. e.g., White III R A,Blainey P C, Fan C H, Quake S R. “Digital PCR provides sensitive andabsolute calibration for high throughput sequencing” BMC Genomics 10:116doi:10.1186/1471-2164-10-116.

Sample Nucleic Acids

Preparations of nucleic acids (“samples”) can be obtained frombiological sources and prepared using conventional methods known in theart. In particular, DNA or RNA useful in the methods described hereincan be extracted and/or amplified from any source, including bacteria,protozoa, fungi, viruses, organelles, as well higher organisms such asplants or animals, particularly mammals, and more particularly humans.Suitable nucleic acids can also be obtained from environmental sources(e.g., pond water), from man-made products (e.g., food), from forensicsamples, and the like. Nucleic acids can be extracted or amplified fromcells, bodily fluids (e.g., blood, a blood fraction, urine, etc.), ortissue samples by any of a variety of standard techniques. Illustrativesamples include samples of plasma, serum, spinal fluid, lymph fluid,peritoneal fluid, pleural fluid, oral fluid, and external sections ofthe skin; samples from the respiratory, intestinal genital, and urinarytracts; samples of tears, saliva, blood cells, stem cells, or tumors.For example, samples of fetal DNA can be obtained from an embryo or frommaternal blood. Samples can be obtained from live or dead organisms orfrom in vitro cultures. Illustrative samples can include single cells,paraffin-embedded tissue samples, and needle biopsies. Nucleic acidsuseful in the invention can also be derived from one or more nucleicacid libraries, including cDNA, cosmid, YAC, BAC, P1, PAC libraries, andthe like.

Nucleic acids of interest can be isolated using methods well known inthe art, with the choice of a specific method depending on the source,the nature of nucleic acid, and similar factors. The sample nucleicacids need not be in pure form, but are typically sufficiently pure toallow the amplification steps of the methods of the invention to beperformed. Where the target nucleic acids are RNA, the RNA can bereversed transcribed into cDNA by standard methods known in the art andas described in Sambrook, J., Fritsch, E. F., and Maniatis, T.,Molecular Cloning: A Laboratory Manual. Cold Spring Harbor LaboratoryPress, NY, Vol. 1, 2, 3 (1989), for example. The cDNA can then beanalyzed according to the methods of the invention.

Target Nucleic Acids

Any target nucleic acid that can be tagged in an encoding reaction ofthe invention (described herein) can be detected using the methods ofthe invention. In typical embodiments, at least some nucleotide sequenceinformation will be known for the target nucleic acids. For example, ifthe encoding reaction employed is PCR, sufficient sequence informationis generally available for each end of a given target nucleic acid topermit design of suitable amplification primers. In an alternativeembodiment, the target-specific sequences in primers could be replacedby random or degenerate nucleotide sequences.

The targets can include, for example, nucleic acids associated withpathogens, such as viruses, bacteria, protozoa, or fungi; RNAs, e.g.,those for which over- or under-expression is indicative of disease,those that are expressed in a tissue- or developmental-specific manner;or those that are induced by particular stimuli; genomic DNA, which canbe analyzed for specific polymorphisms (such as SNPs), alleles, orhaplotypes, e.g., in genotyping. Of particular interest are genomic DNAsthat are altered (e.g., amplified, deleted, and/or mutated) in geneticdiseases or other pathologies; sequences that are associated withdesirable or undesirable traits; and/or sequences that uniquely identifyan individual (e.g., in forensic or paternity determinations).

Primer Design

Primers suitable for nucleic acid amplification are sufficiently long toprime the synthesis of extension products in the presence of the agentfor polymerization. The exact length and composition of the primer willdepend on many factors, including, for example, temperature of theannealing reaction, source and composition of the primer, and where aprobe is employed, proximity of the probe annealing site to the primerannealing site and ratio of primer:probe concentration. For example,depending on the complexity of the target nucleic acid sequence, anoligonucleotide primer typically contains in the range of about 15 toabout 30 nucleotides, although it may contain more or fewer nucleotides.The primers should be sufficiently complementary to selectively annealto their respective strands and form stable duplexes. One skilled in theart knows how to select appropriate primer pairs to amplify the targetnucleic acid of interest.

For example, PCR primers can be designed by using any commerciallyavailable software or open source software, such as Primer3 (see, e.g.,Rozen and Skaletsky (2000) Meth. Mol. Biol., 132: 365-386;www.broad.mit.edu/node/1060, and the like) or by accessing the Roche UPLwebsite. The amplicon sequences are input into the Primer3 program withthe UPL probe sequences in brackets to ensure that the Primer3 programwill design primers on either side of the bracketed probe sequence.

Primers may be prepared by any suitable method, including, for example,cloning and restriction of appropriate sequences or direct chemicalsynthesis by methods such as the phosphotriester method of Narang et al.(1979) Meth. Enzymol. 68: 90-99; the phosphodiester method of Brown etal. (1979) Meth. Enzymol. 68: 109-151; the diethylphosphoramidite methodof Beaucage et al. (1981) Tetra. Lett., 22: 1859-1862; the solid supportmethod of U.S. Pat. No. 4,458,066 and the like, or can be provided froma commercial source.

Primers may be purified by using a Sephadex column (AmershamBiosciences, Inc., Piscataway, N.J.) or other methods known to thoseskilled in the art. Primer purification may improve the sensitivity ofthe methods of the invention.

Microfluidic Devices

In certain embodiments, any of the methods of the invention can becarried out using a microfluidic device. In illustrative embodiments,the device is a matrix-type microfluidic device is one that allows thesimultaneous combination of a plurality of substrate solutions withreagent solutions in separate isolated reaction chambers. It will berecognized, that a substrate solution can comprise one or a plurality ofsubstrates and a reagent solution can comprise one or a plurality ofreagents. For example, the microfluidic device can allow thesimultaneous pair-wise combination of a plurality of differentamplification primers and samples. In certain embodiments, the device isconfigured to contain a different combination of primers and samples ineach of the different chambers. In various embodiments, the number ofseparate reaction chambers can be greater than 50, usually greater than100, more often greater than 500, even more often greater than 1000, andsometimes greater than 5000, or greater than 10,000.

In particular embodiments, the matrix-type microfluidic device is aDynamic Array (“DA”) microfluidic device, an example of which is shownin FIG. 1. A DA microfluidic device is a matrix-type microfluidic devicedesigned to isolate pair-wise combinations of samples and reagents(e.g., amplification primers, detection probes, etc.) and suited forcarrying out qualitative and quantitative PCR reactions includingreal-time quantitative PCR analysis. In some embodiments, the DAmicrofluidic device is fabricated, at least in part, from an elastomer.DA microfluidic devices are described in PCT publication WO05107938A2(Thermal Reaction Device and Method For Using The Same) and US Pat.Publication US20050252773A1, both incorporated herein by reference intheir entireties for their descriptions of DA microfluidic devices. DAmicrofluidic devices may incorporate high-density matrix designs thatutilize fluid communication vias between layers of the microfluidicdevice to weave control lines and fluid lines through the device andbetween layers. By virtue of fluid lines in multiple layers of anelastomeric block, high density reaction cell arrangements are possible.Alternatively DA microfluidic devices may be designed so that all of thereagent and sample channels are in the same elastomeric layer, withcontrol channels in a different layer.

U.S. Patent Publication No. 2008/0223721 and PCT Publication No. WO05/107938A2 describe illustrative matrix-type devices that can be usedto practice the methods described herein. FIG. 21 of the latter isreproduced as FIG. 1 and shows an illustrative matrix design having afirst elastomeric layer 2110 (1st layer) and a second elastomeric layer2120 (2d layer) each having fluid channels formed therein. For example,a reagent fluid channel in the first layer 2110 is connected to areagent fluid channel in the second layer 2120 through a via 2130, whilethe second layer 2120 also has sample channels therein, the samplechannels and the reagent channels terminating in sample and reagentchambers 2180, respectively. The sample and reagent chambers 2180 are influid communication with each other through an interface channel 2150that has an interface valve 2140 associated therewith to control fluidcommunication between each of the chambers 2180 of a reaction cell 2160.In use, the interface is first closed, then reagent is introduced intothe reagent channel from the reagent inlet and sample is introduced intothe sample channel through the sample inlet; containment valves 2170 arethen closed to isolate each reaction cell 2160 from other reaction cells2160. Once the reaction cells 2160 are isolated, the interface valve2140 is opened to cause the sample chamber and the reagent chamber to bein fluid communication with each other so that a desired reaction maytake place. It will be apparent from this (and the description in WO05/107938A2) that the DA microfluidic device may be used for reacting Mnumber of different samples with N number of different reagents.

Although the DA microfluidic devices described above in WO 05/107938 arewell suited for conducting the methods described herein, the inventionis not limited to any particular device or design. Any device thatpartitions a sample and/or allows independent pair-wise combinations ofreagents and sample may be used. U.S. Patent Publication No. 20080108063(which is hereby incorporated by reference it its entirety) includes adiagram illustrating the 48.48 Dynamic Array IFC (Integrated FluidicCircuit), a commercially available device available from Fluidigm Corp.(South San Francisco Calif.). It will be understood that otherconfigurations are possible and contemplated such as, for example,48×96; 96×96; 30×120; etc.

In specific embodiments, the microfluidic device can be a Digital Arraymicrofluidic device, which is adapted to perform digital amplification.Such devices can have integrated channels and valves that partitionmixtures of sample and reagents into nanolitre volume reaction chambers.In some embodiments, the Digital Array microfluidic device isfabricated, at least in part, from an elastomer. Illustrative DigitalArray microfluidic devices are described in copending U.S. applicationsowned by Fluidigm, Inc., such as U.S. application Ser. No. 12/170,414,entitled “Method and Apparatus for Determining Copy Number VariationUsing Digital PCR.” One illustrative embodiment has 12 input portscorresponding to 12 separate sample inputs to the device. The device canhave 12 panels, and each of the 12 panels can contain 765 6 nL reactionchambers with a total volume of 4.59 μL per panel. Microfluidic channelscan connect the various reaction chambers on the panels to fluidsources. Pressure can be applied to an accumulator in order to open andclose valves connecting the reaction chambers to fluid sources. Inillustrative embodiments, 12 inlets can be provided for loading of thesample reagent mixture. 48 inlets can be used to provide a source forreagents, which are supplied to the biochip when pressure is applied toaccumulator. Additionally, two or more inlets can be provided to providehydration to the biochip. Hydration inlets are in fluid communicationwith the device to facilitate the control of humidity associated withthe reaction chambers. As will be understood to one of skill in the art,some elastomeric materials that can utilized in the fabrication of thedevice are gas permeable, allowing evaporated gases or vapor from thereaction chambers to pass through the elastomeric material into thesurrounding atmosphere. In a particular embodiment, fluid lines locatedat peripheral portions of the device provide a shield of hydrationliquid, for example, a buffer or master mix, at peripheral portions ofthe biochip surrounding the panels of reaction chambers, thus reducingor preventing evaporation of liquids present in the reaction chambers.Thus, humidity at peripheral portions of the device can be increased byadding a volatile liquid, for example water, to hydration inlets. In aspecific embodiment, a first inlet is in fluid communication with thehydration fluid lines surrounding the panels on a first side of thebiochip and the second inlet is in fluid communication with thehydration fluid lines surrounding the panels on the other side of thebiochip.

While the Digital Array microfluidic devices are well-suited forcarrying out the digital amplification methods described herein, one ofordinary skill in the art would recognize many variations andalternatives to these devices. The microfluidic device which is the12.765 Dynamic Array commercially available from Fluidigm Corp. (SouthSan Francisco, Calif.), includes 12 panels, each having 765 reactionchambers with a volume of 6 nL per reaction chamber. However, thisgeometry is not required for the digital amplification methods describedherein. The geometry of a given Digital Array microfluidic device willdepend on the particular application. Additional description related todevices suitable for use in the methods described herein is provided inU.S. Patent Application Publication No. 2005/0252773, incorporatedherein by reference for its disclosure of Digital Array microfluidicdevices.

In certain embodiments, the methods described herein can be performedusing a microfluidic device that provides for recovery of reactionproducts. Such devices are described in detail in copending U.S.Application No. 61/166,105, filed Apr. 2, 2009, which is herebyincorporated by reference in its entirety and specifically for itsdescription of microfluidic devices that permit reaction productrecovery and related methods. For example, the digital PCR method forcalibrating DNA samples prior to sequencing can be preformed on suchdevices, permitting recovery of amplification products, which can thenserve as templates for DNA sequencing.

FIG. 2 is a simplified perspective illustration of a carrier and amicrofluidic device according to an embodiment of the present invention.As illustrated in FIG. 2, the carrier 100 supports a microfluidic device110, which may also be referred to as a Digital Array microfluidicdevice. The carrier 100 may be made from materials providing suitablemechanical support for the various elements of the carrier. As anexample, the carrier is made using a plastic or other suitable material.The outer portion of the carrier has the same footprint as a standard384-well microplate and enables stand-alone valve operation.Additionally, the carrier 100 is compatible with conventionalstand-alone thermal cyclers. As described below, there are 48 sampleinput ports 120 located on a first side of the carrier 100 and 48 assayinput ports 122 located on an opposing side of the carrier. The banks ofsample input ports 120 and assay input ports 122 are recessed withrespect to the top of the carrier. Utilizing these recessed features,pressure can be applied concurrently to all of the sample input ports orthe assay input ports, driving fluids present in the respective portsthrough fluid lines 140 connecting the input ports and either vias,fluid input lines, or combinations thereof, present on the microfluidicdevice 110. The samples may include encoded primers and the assays mayalso be referred to as amplicon-specific (AS) primers.

The carrier 100 also includes four sources 130, 132, 134, and 136, whichmay be used to actuate control lines present in the microfluidic device.In an embodiment, sources 130, 132, and 134 are used to pressurizecontrol lines operable to open and close valves present in themicrofluidic device. For example, application of pressure greater thanatmospheric pressure to source 132 will result in the liquid present insource 132 flowing into control lines present on the microfluidicdevice, thereby actuating valves operable to obstruct flow through oneor more fluid input lines also present on the microfluidic device. In anembodiment, source 130 is used as a fluid well containing harvestingreagent. Pressure can be applied to source 130, forcing the harvestingreagent to flow through fluid lines provided on the carrier to fluidlines provided on the microfluidic device. Thus, application of pressureto source 130 can result in the flow of a harvesting reagent or othersuitable fluid through the microfluidic device. The control lines thatare in fluid communication with the sources 130-136 can include controllines for interface valves, containment valves, valves used in dilationpumping, fluid lines for the flow of harvesting reagent, or the like. Ina particular embodiment, valve 1 is controlled by source 132, valve 2 iscontrolled by source 134, harvesting reagent is provided in source 130,and hydration reagent is provided in source 136. In this particularembodiment, the interface valves are controlled by source 150 andcontainment valves are controlled by source 152. This particularembodiment is not intended to limit the present invention, but merely toprovide an example of one configuration. Other configurations can beutilized as appropriate to the particular application.

As described more fully in relation to FIG. 3, fluid lines 140 presenton the carrier 100 are in fluid communication with one or more fluidlines present on the microfluidic device 110. These fluid lines canserve to carry fluids into and out of the microfluidic device or may beused as control lines to actuate valves present on the microfluidicdevice. Thus, fluids provided in sample input ports 120 or assay inputports 122 can be loaded into appropriate fluid input lines and chambersof the microfluidic device. Other fluids (e.g., liquids) provided insources 130-136 can also be loaded into either fluid input lines orcontrol lines of the microfluidic device. Reaction products from thechambers of the microfluidic device can be recovered as they are pumpedthrough fluid lines on the microfluidic device, back into the fluidlines 140 present on the carrier and into the sample or assay inputports 120 or 122.

Pressure accumulators 150 and 152 may be utilized to pressurize othercontrol lines, provide for hydration of the microfluidic device, or theymay not be used in some embodiments. Although 48 sample input ports and48 assay input ports are shown in the embodiment of the presentinvention illustrated in FIG. 2, this is not required by the presentinvention. Other embodiments utilize a different number of samples andassays depending on the particular application. One of ordinary skill inthe art would recognize many variations, modifications, andalternatives.

FIG. 3 is a simplified schematic diagram of a microfluidic deviceaccording to an embodiment of the present invention. The microfluidicdevice 200 includes vias 210 connected to fluid input lines 212 that areused to provide fluid flow paths for 24 different samples. The 24samples, which can be loaded into a subset of the sample input ports 120illustrated in FIG. 1, flow through vias 210 and fluid input lines 212to sample input lines 312 and eventually to sample chambers 310 asillustrated in FIG. 4. The microfluidic device 200 also includes vias220 connected to fluid input lines 222 that are used to provide fluidflow paths for 21 different assays. The via 250 on the side of themicrofluidic device opposing the assay input fluid lines provides forhydration, actuation of a control line, or other suitable operations.The array portion 230 of the microfluidic device is illustrated (inpart) in FIG. 4. In the array portion 230, the samples and assays areloaded into sample and assay chambers and then can be mixed to formpairwise combinations.

As described more fully throughout the present specification, aftersamples and assays are mixed and reacted, the reaction products can berecovered from the microfluidic device by flowing a recovery fluidthrough the fluid input lines 212, through the array portion 230 of themicrofluidic device as illustrated in FIG. 4, and through the outputfluid lines 240 and vias 242. These output fluid lines are in fluidcommunication with output ports provided on a carrier. Thus, reactionproducts pooled from the combination of a sample with each of thevarious assays are separately provided through each of the independentoutput fluid lines 240.

The particular number of sample and assay input lines illustrated inFIG. 3 are provided merely by way of example and particularimplementations are not limited to these particular numbers. In otherembodiments, additional sample and assay input lines are provided inorder to facilitate additional pairwise combinations of samples andassays in the microfluidic device.

FIG. 4 is a simplified schematic diagram of several unit cells of themicrofluidic device illustrated in FIG. 3. In FIG. 4, four unit cellsfrom the array portion 230 are illustrated for purposes of clarity. Thesample input lines 316 are in fluid communication with the fluid inputlines 212 and the assay input lines 318 are in fluid communication withthe assay input lines 222 as illustrated in FIG. 3. The unit cellsection of the microfluidic device includes sample chambers 310 andassay chambers 312. Fluid lines 314 provide for fluid communicationbetween the sample chambers and the assay chambers when the interfacevalves 330 are in the open position. In a specific embodiment, sampleinput lines 316 are provided in a layer of the microfluidic deviceunderlying the layer containing the sample chambers. In a similarmanner, assay input lines 318 are provided in a layer of themicrofluidic device underlying the layer containing the assay chambers.Samples flow from the sample input lines 212 illustrated in FIG. 3 tosample input lines 316 and up through one or more vias (not shown)passing from the sample input lines 316 to the sample chambers 310.Although the sample input lines 316 are illustrated as branching intothree input lines as the fluid passes to the sample chambers, thisparticular number is not required by the present invention and othernumbers of sample input lines, for example, 1 input line, 2, 4, or morethan 4 sample input lines may be utilized. Similar design criteria areapplicable to the three fluid lines 314 connecting the sample chambersand corresponding assay chambers. The sample input lines 316 provide acontinuous flow path in the row direction of the figure, enabling asingle sample to be distributed evenly among multiple sample chambers,for example, the top row of sample chambers or the bottom row of samplechambers.

Utilizing interface valves 330 and containment valves 340, each of thesample chambers can be isolated from each of the other sample chambersas well as the assay chambers. The assay chambers can be isolated fromthe other assay chambers using the containment valves. Both theisolation and containment valves are actuated by application of pressureto a corresponding control line present on the carrier or by othermeans, for example, electrostatic actuation.

FIG. 4 illustrates assay input lines 318, which provide for assay flowfrom assay input lines 222 illustrated in FIG. 3 to the assay chambers312. When the containment valves are in the open position, assays areable to flow from the assay input lines to the assay chambers 312. In aspecific embodiment, the assays flow through vias connecting the inputlines and the assay chambers in a manner similar to the filling of thesample chambers. The loading of assays along the columns of themicrofluidic device provide a different assay for each row of samples,resulting in M×N pairwise combinations.

Opening of the interface valves 330 enables the samples and the assaysto mix in pairwise combinations via free interface diffusion. After thesamples and assays are mixed, thermocycling can be performed to formreaction products. Reaction products are recovered from the microfluidicdevice by opening harvest valves 350, which enable the reaction productsto flow into portions 360 of the sample input lines adjacent the samplechambers. Using sample input lines 316 and on-chip pumps (not shown),reaction products flow through the sample input lines toward recoveryports on the carrier.

In the embodiment illustrated in FIG. 4, samples load from a first sideof the microfluidic device. The assays load from an adjacent side of themicrofluidic device. After processing, a harvesting reagent is inputfrom the first side of the device using the sample input lines andreaction products exit the microfluidic device out fluid lines runningtoward the side of the microfluidic device opposing the first side. Inthis embodiment, the remaining side of the microfluidic device is notused for sample or assay loading or reaction product unloading. Otherconfigurations are included within the scope of the present inventionand the example configuration illustrated in FIG. 4 is merely providedby way of example. One of ordinary skill in the art would recognize manyvariations, modifications, and alternatives.

A benefit provided by the systems described herein is that the volume ofsamples and assays used in the reactions are fixed, regardless of thepipetting volume dispensed into the sample input and the assay inputports. If the volume in the sample and/or assay input ports is above apredetermined threshold sufficient to fill the sample/assay input linesand the sample/assay chambers, then application of pressure to thesample/assay input ports will result in complete filling of thesample/assay chambers. The completely filled chambers thus provide afixed reaction volume not available with conventional microtiter platetechniques.

Although systems have been developed by the present assignee to performmany simultaneous binding assays, including, but not limited toimmunological experiments such as ELISA assays, embodiments of thepresent invention provide for dilation pumping “on-chip” as well asseparate sample and assay chambers. Thus, pairwise combinations ofsamples and assays are possible using embodiments described herein thatare not possible with previously developed techniques. Additionaldescription of binding assays is provided in U.S. Patent ApplicationPublication No. 2007/0074972, filed on Sep. 13, 2006, the disclosure ofwhich is hereby incorporated by reference in its entirety for allpurposes.

Embodiments of the present invention provide a system suitable for PCRsample preparation that features reduced cost, time, and labor in thepreparation of amplicon libraries from an input DNA template. In atypical use case, the first amplification will be used to generatelibraries for next-generation sequencing. Utilizing embodiments of thepresent invention, samples and encoded primers are combined withamplicon-specific (AS) primers to create a mixture that is suitable fordesired reactions. Based on an M×N architecture of the microfluidicdevice, each of the M samples is combined with each of the N AS primers(i.e., assays) to form M×N pairwise combinations. That is, one reactionsite is provided for each sample and assay pair. After the completion ofthe reaction (e.g., PCR), the reaction products are recovered from thesystem, typically using a harvest reagent that flows through themicrofluidic device. In a specific embodiment, reaction productsassociated with each sample are recovered in a separate reaction pool,enabling further processing or study of the pool containing a givensample reacted with each of the various assays.

Thus, in embodiments described herein, a microfluidic device is providedin which independent sample inputs are combined with primer inputs in anM×N array configuration. Thus, each reaction is a unique combination ofa particular sample and a particular primer. As described more fullythroughout the present specification, samples are loaded into samplechambers in the microfluidic device through sample input lines arrangedas columns in one implementation. AS primers or assays are loaded intoassay chambers in the microfluidic device through assay input linesarranged as rows crossing the columns. The sample chambers and the assaychambers are in fluidic isolation during loading. After the loadingprocess is completed, an interface valve operable to obstruct a fluidline passing between pairs of sample and assay chambers is opened toenable free interface diffusion of the pairwise combinations of samplesand assays. Precise mixture of the samples and assays enables reactionsto occur between the various pairwise combinations, producing a reactionproduct including a set of specific PCR reactions for which each samplehas been effectively coded with a unique barcode. The reaction productsare harvested and can then be used for subsequent sequencing processes.The terms “assay” and “sample” as used herein are descriptive ofparticular uses of the devices in some embodiments. However, the uses ofthe devices are not limited to the use of “sample(s)” and “assay(s)” inall embodiments. For example, in other embodiments, “sample(s)” mayrefer to “a first reagent” or a plurality of “first reagents” and“assay(s)” may refer to “a second reagent” or a plurality of “secondreagents.” The M×N character of the devices enable the combination ofany set of first reagents to be combined with any set of secondreagents.

According to one particular process implemented using an embodiment ofthe present invention, after 25 cycles of PCR, the reaction productsfrom the M×N pairwise combinations will be recovered from themicrofluidic device in discrete pools, one for each of the M samples.Typically, the discrete pools are contained in a sample input portprovided on the carrier. In some processes, the reaction products may beharvested on a “per amplicon” basis for purposes of normalization.Utilizing embodiments of the present invention, it is possible toachieve results (for replicate experiments assembled from the same inputsolutions of samples and assays) for which the copy number ofamplification products varies by no more than ±25% within a sample andno more than ±25% between samples. Thus, the amplification productsrecovered from the microfluidic device will be representative of theinput samples as measured by the distribution of specific knowngenotypes. Preferably, output sample concentration will be greater than2,000 copies/amplicon/microliter and recovery of reaction products willbe performed in less than two hours.

Applications in which embodiments of the present invention can be usedinclude sequencer-ready amplicon preparation and long-range PCR ampliconlibrary production. For the sequencer-ready amplicon preparation,multiple-forward primer and 3-primer combination protocols can beutilized.

FIG. 5A is simplified schematic diagram of a microfluidic deviceaccording to another embodiment of the present invention. Themicrofluidic device illustrated in FIG. 5A shares common features aswell as differences with the microfluidic device illustrated in FIG. 2.Samples are loaded into the microfluidic device through 48 vias andcorresponding sample input lines provided at one edge of themicrofluidic device (i.e., the bottom edge in FIG. 5A). Samples flowthrough the sample input lines into the array portion 430 of themicrofluidic device. The assays are loaded from vias and assay inputlines on two sides of the microfluidic device (i.e., the left and rightsides in FIG. 5A). Additional discussion of the unit cells present inthe array portion 430 is provided in relation to FIG. 6. Reactionproducts are removed through the sample input lines and are recovered inthe sample input ports 120 provided on the carrier 100. Thus, in FIG.5A, loading of samples and recovery of reaction products are illustratedas flowing through the bottom side of the microfluidic device.

FIG. 5B is a simplified schematic diagram of portions of themicrofluidic device illustrated in FIG. 5A. The portions illustrated inFIG. 5B include sample input lines 410, assay input lines for evenassays (assay input lines 420), assay input lines for odd assays (assayinput lines 422), and a harvesting reagent input lines 430. In anembodiment, the sample input lines 410 are in fluid communication withvias 412 that are aligned with sample input lines 140, which are influid communication with sample input ports 120 as illustrated in FIG.2. In other embodiments, additional sample input lines (not shown) areprovided to enable fluid communication between the sample input ports120 and the sample input lines 410. Thus, pressurization of the bank ofsample input ports will result in flow of the various samples into theillustrated sample input lines 410.

As discussed in relation to FIG. 6 below, sample input lines 410 are influid communication with sample input lines 516 and sample chambers 510present in the microfluidic device. For an array with 48 sample chambersand 48 assay chambers, the 48 sample input lines 410 illustrated in FIG.5B will provide samples to 48 separate columns of sample chambers, twoof which are illustrated in FIG. 6. It should be noted that the variousfluid lines illustrated in FIG. 5B can be integrated in carrier 100,integrated in the microfluidic device 110, or present in one or moreother structures, depending on the particular implementation. Thus, theillustration of the sample input lines 410 in FIG. 5B is not intended tolimit the scope of the present invention but merely to illustrate fluidlines suitable for providing controlled fluid flow to the variouschambers of the microfluidic device.

In an embodiment, even assay input lines 420 and odd assay input lines422 are in fluid communication with vias 424 that are aligned with assayinput lines 140, which are in fluid communication with assay input ports122 as illustrated in FIG. 2. In other embodiments, additional assayinput lines (not shown) are provided to enable fluid communicationbetween the assay input ports 122 and the assay input lines 420 and 422.Thus, pressurization of the bank of assay input ports 122 will result inflow of the various assays into the illustrated assay input lines. Afterflowing through the input lines illustrated in FIG. 5B, the variousfluids will eventually enter into the unit cells illustrated in FIG. 6.

As discussed above, the various fluid lines can be integrated into thecarrier, the microfluidic device, or other suitable structure. In a 48sample×48 assay array configuration, the 24 even assay input lines 420will provide inputs to half of the rows of assay input lines 518 shownin FIG. 6. The 24 odd assay input lines 422 will provide inputs to halfof the rows of assay inputs lines 518 shown in FIG. 6. Thus, althoughloading of assays is only illustrated from the right side of the arrayin FIG. 6, actual implementation will typically load assays from bothsides in an even/odd configuration. In some embodiments, additional viasare provide for loading of hydration fluids or the like. Moreover, insome embodiments, in order to provide compatibility with existingcarriers, some fluid lines are unused or modified to provide for suchcompatibility.

The harvesting reagent input line 430 provides for harvesting reagentused in recovering reaction products from the microfluidic device. Theharvesting reagent input line 430 illustrated in FIG. 5B is in fluidcommunication with the harvesting reagent input port 136 illustrated inFIG. 2 and passes along the microfluidic device adjacent to the evenassay input lines to the top portion of the device and then branches offinto a plurality of harvesting reagent input lines. The harvestingreagent multiplexor has a substantially equal volume for every sampleinput line to provide uniform pumping during the reaction productrecovery operation. It should be noted that the particular branchingsystem illustrated in FIG. 5B is merely provided as an example and otherbranching systems are included within the scope of the presentinvention. The harvesting reagent input lines 430 are in fluidcommunication with sample input lines 516 discussed in relation to FIG.6. As discussed in relation to FIGS. 9A-D, embodiments utilize aseparate harvesting reagent input line for each column of samplechambers, for example, 48 harvesting reagent input lines for anmicrofluidic device with a 48×48 array configuration. Additionally,although the harvesting reagent input line enters the microfluidicdevice at a location adjacent the even assay input lines 420, this isnot required by embodiments of the present invention and otherconfigurations are within the scope of the present invention. One ofordinary skill in the art would recognize many variations,modifications, and alternatives.

As described in relation to FIG. 8C, harvesting reagent flows from aharvesting reagent input port on the carrier, through the harvestingreagent input lines 430 and into one end of the sample input lines 516.As discussed more fully throughout the present specification, the sampleinput lines function both as input lines and reaction product recoverylines. For loading, the sample flow path is from the sample input ports120, through input lines 140, through vias 412, through sample inputlines 410, through sample input lines 516, to the reaction chambers 510and to the loading bowls 830. For reaction product recovery, the productflow path is from the harvesting reagent input port 136, through theharvesting reagent input lines 430, through the sample input lines 516,through the sample input lines 410, through the vias 412, and to thesample input ports 120, which serve during harvesting, as a fluidrecovery well. Thus, the use of the term “input” lines should beconsidered in the context of the particular process being performed,since the “input” lines can serve to both load samples and recover orremove reaction products from the microfluidic device and the carrier.

By applying pressure to the bank of sample input ports 120 and the bankof assay input ports 122, samples and reagents can be loaded through theillustrated sample and assay input lines into sample and assay chamberspresent in the microfluidic device. By applying pressure to theharvesting reagent input port 136, the reaction products can berecovered from the sample chambers and delivered to the sample inputports. Valves present in the microfluidic device are utilized to controlthe flow of samples, assays, and reaction products, as described morefully throughout the present specification. FIG. 5B only illustrates aportion of the sample input lines, assay input lines, and harvestingreagent input lines and additional portions of these input lines areillustrated in FIG. 5A and FIG. 6.

FIG. 6 is a simplified schematic diagram of several unit cells of themicrofluidic device illustrated in FIG. 5A. In FIG. 6, four unit cellsfrom the array portion 430 shown in FIG. 5A are illustrated for purposesof clarity. The unit cell section of the microfluidic device includessample chambers 510 and assay chambers 512. Fluid lines 514 provide forfluid communication between the sample chambers and the assay chamberswhen the interface valves 530 are in the open position. In a specificembodiment, sample input lines 516 are provided in a layer of themicrofluidic device underlying the layer containing the sample chambers.In a similar manner, assay input lines 518 are provided in a layer ofthe microfluidic device underlying the layer containing the assaychambers. Samples flow from the sample input lines 410 illustrated inFIG. 5B to sample input lines 516 and up through one or more viaspassing from the sample input lines to the sample chambers. Although twosample lines are illustrated for each sample chambers, this particularnumber is not required by the present invention and other numbers ofsample input lines, for example, 1 input line, 3, 4, or more than 4sample input lines may be utilized. The sample input lines 516 provide acontinuous flow path in the column direction of the figure, enabling asingle sample to be distributed evenly among multiple sample chambers.

FIG. 6 illustrates assay input lines 518, which provide for assay flowfrom assay input lines 420 and 422 illustrated in FIG. 5B to the assaychambers 512. Although FIG. 6 only illustrates assay input linesentering the unit cells from the right side of the figure, it will beevident to one of skill in the art that in the implementationillustrated in FIG. 5B, even and odd assays are loaded from opposingsides of the microfluidic device. The illustration provided in FIG. 6 ismerely simplified for purposes of clarity. In a specific embodiment, theassays load through vias connecting the input lines and the assaychambers in a manner similar to the filling of the sample chambers. Theloading of assays along the rows of the microfluidic device provide adifferent assay for each of the samples, resulting in a number ofpairwise combinations appropriate for an M×N array.

As described more fully throughout the present specification, reactionproducts are recovered from the microfluidic device using the sampleinput lines 516 and pumps (not shown). Containment valves 540 providefor containment between the various sample and assay chambers in eachrow. Utilizing the interface valves 530 and the containment valves 540,each of the sample chambers can be isolated from each of the othersample chambers as well as the assay chambers. The assay chambers can beisolated from the other assay chambers using the containment valves.Both the isolation and containment valves are actuated by application ofpressure to a corresponding control line in fluid communication withsources 130-134 or by other means, for example, electrostatic actuation.

In FIG. 6, four sample chambers 510 are illustrated in an arrayconfiguration. The four illustrated chambers are merely shown by way ofexample and implementations of the present invention are not limited tothe four illustrated chambers, but typically provide 2,304 chambers in a48×48 array configuration, 4,096 chambers in a 64×64 arrayconfiguration, 9,216 chambers in a 96×96 array configuration, or thelike.

Embodiments of the present invention provide unit cells with dimensionson the order of several hundred microns, for example unit cells withdimension of 500×500 μm, 525×525 μm, 550×550 μm, 575×575 μm, 600×600 μm,625×625 μm, 650×650 μm, 675×675, μm, 700×700 μm, or the like. Thedimensions of the sample chambers and the assay chambers are selected toprovide amounts of materials sufficient for desired processes whilereducing sample and assay usage. As examples, sample chambers can havedimensions on the order of 100-400 μm in width×200-600 μm inlength×100-500 μm in height. For example, the width can be 100 μm, 125μm, 150 μm, 175 μm, 200 μm, 225 μm, 250 μm, 275 μm, 300 μm, 325 μm, 350μm, 375 μm, 400 μm, or the like. For example, the length can be 200 μm,225 μm, 250 μm, 275 μm, 300 μm, 325 μm, 350 μm, 375 μm, 400 μm, 425 μm,450 μm, 475 μm, 500 μm, 525 μm, 550 μm, 575 μm, 600 μm, or the like. Forexample, the height can be 100 μm, 125 μm, 150 μm, 175 μm, 200 μm, 225μm, 250 μm, 275 μm, 300 μm, 325 μm, 350 μm, 375 μm, 400 μm, 425 μm, 450μm, 475 μm, 500 μm, 525 μm, 550 μm, 575 μm, 600 μm, or the like. Assaychambers can have similar dimensional ranges, typically providingsimilar steps sizes over smaller ranges than the smaller chambervolumes. In some embodiments, the ratio of the sample chamber volume tothe assay chamber volume is about 5:1, 10:1, 15:1, 20:1, 25:1, or 30:1.Smaller chamber volumes than the listed ranges are included within thescope of the invention and are readily fabricated using microfluidicdevice fabrication techniques.

Higher density microfluidic devices will typically utilize smallerchamber volumes in order to reduce the footprint of the unit cells. Inapplications for which very small sample sizes are available, reducedchamber volumes will facilitate testing of such small samples.

The dimensions of the interface valves 530 are selected to provide forcomplete obstruction of the fluid lines 514 connecting the sample andassay chambers. In some embodiments, the valve dimensions range fromabout 10-200 μm×10-200 μm, for example, 50×50 μm, 50×65 μm, 50×80 μm,50×100 μm, 65×50 μm, 65×65 μm, 65×80 μm, 65×100 μm, 80×50 μm, 80×65 μm,80×80 μm, 80×100 μm, 100×50 μm, 100×65 μm, 100×80 μm, 100×100 μm, or thelike. The sample input lines may have various widths depending on thenumber of sample input lines and the sample chamber volumes, and desiredflow rates for loading and product recovery. As examples, the sampleinput lines may have a cross-section of 1-20 μm in height and 50-100 μmin width. For example, the sample input lines may have heights of 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μmand widths of 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 μm.

Other device parameters, including layer to layer alignment, rangingfrom 20-100 μm, and via size, ranging from 50-200 microns, are selectedto provide desired system performance characteristics. One of ordinaryskill in the art would recognize many variations, modifications, andalternatives.

In some embodiments, an extra assay inlet is provided at the side of themicrofluidic device adjacent the harvesting reagent input lines.Additionally, no assay inlet is provided at the side of the microfluidicdevice adjacent the sample input ports on the carrier. In thisconfiguration, the extra assay inlet can be used for dehydration chamberloading. Typically, loading of the dehydration chambers will use morethan 5 μl of assay solution. Alternatively, a separate dehydrationsolution could be used to keep assay volumes uniform across themicrofluidic device.

FIG. 7 is a simplified flowchart of a method of operating a microfluidicdevice according to an embodiment of the present invention. In theillustrated embodiment, the microfluidic device includes at least oneassay chamber, at least one sample chamber, and at least one harvestingport. In a particular embodiment, the microfluidic device includes aplurality of assay chambers and a plurality of sample chambers. Themethod 600 includes closing a fluid line between the assay chamber andthe sample chamber (610). Referring to FIG. 6, interface valves 530 areoperable to close fluid lines 514 passing between the sample chambers510 to the assay chambers 512. In some embodiments, the interface valves530 are “push-up” valves as described more fully below. The interfacevalves are formed by the intersection of control line 532 or controlchannel that is at least partially contained in a first layer of themicrofluidic device. The fluid lines are at least partially contained ina second layer of the microfluidic device. The control lines 532 are influid communication with one or more pressure actuators or accumulatorsas illustrated in FIG. 2.

The intersection of the control line 532 with the fluid line 514 forms avalve at the intersection, referred to as an interface valve 530 becausethe valve prevents mixing at the interface between the sample and theassay. The interface valve 530 is actuated in response to fluid pressurein the control line and is operative to prevent fluid flow through thefluid lines. Generally, the multilayer microfluidic device discussedherein includes a number of elastomeric layers and the valves 530include a deflectable membrane between the first layer and the secondlayer. In a “push-up” configuration, the deflectable membrane of thevalve is deflectable into the fluid line 514 positioned above theintersection with the control line 532. In this configuration, thedeflectable membrane deflects up into the fluid line to close the fluidline at the position of the valve, thus the reference to “push-up”valves. Releasing the pressure in the control line will result in thedeflectable membrane returning to the undeflected position and therebyopening of the closed valve. Additional description of microfluidicdevices including valves is provided in U.S. Patent Application No.2005/0226742, the entire disclosure of which is hereby incorporated byreference in its entirety for all purposes.

As illustrated in FIG. 6, actuation of control lines 532 will obstructfluid lines 514. Typically, the control lines 532 are actuatedconcurrently by application of pressure to a pressure accumulator.Referring once again to FIG. 7, after closing of the interface valves530, samples flow into sample chambers 510 via sample input lines 516(612). As illustrated in FIG. 6, each sample chamber 510 is in fluidcommunication with multiple (e.g., two) sample input lines 516. In otherembodiments, other numbers of sample input lines can be utilized. One ofordinary skill in the art would recognize many variations,modifications, and alternatives. Typically, the sample input lines,which are at least partially contained in a second layer of themicrofluidic device, pass under the sample chambers, which are at leastpartially contained in a third layer of the microfluidic device. A via(not illustrated) passing from the sample input line up to the samplechamber, provide for fluid flow from the sample input line to the samplechamber. As shown in FIG. 6, samples flow in, for example, up thecolumns, past containment valves 540, which are open, through the viasextending out of the plane of the figure, and into the various samplechambers. Fluids such as air present in the sample chambers are expelledduring loading of the samples as a result of the permeability of theelastomeric material used to fabricate the microfluidic device.

Referring once again to FIG. 7, assays flow into assay chambers 512 viaassay input lines 518 (614). The assays flow through assay input lines518, past containment valves 540, which are open, and through vias (notshown) passing from the assay input lines to the assay chambers. Theclosure of the interface valves 530 prevent the samples in the samplechambers and the assays in the assay chambers from mixing. Once thesample and assay are loaded, the fluid line between the assay chamberand the sample chamber is opened (616). In embodiments of the presentinvention, multiple fluid lines 514 are opened concurrently by openingof interface valves 530. At least a portion of the sample and at least aportion of the assay are combined to form a mixture (618). The mixtureof the sample and assay is formed throughout the sample and assaychambers as well as the fluid lines connecting these chambers. Freeinterface diffusion is a process in which mixing is slow and the rate ofspecies equilibration depends on the species' diffusion constants. Smallmolecules such as salts have large diffusion constants, and henceequilibrate quickly. Large molecules (e.g., proteins) have smalldiffusion constants, and equilibrate more slowly.

The mixture is reacted to form a reaction product (620). A typicalreaction included within the scope of the present invention is PCR,which involves thermocycling of the microfluidic device through a numberof cycles as will be evident to one of skill in the art. The fluid linebetween the assay chamber and the sample chamber is closed (622) byactuation of interface valves 530. Closure of the interface valvesseparates the reaction product present in the sample chambers from thereaction products present in the assay chambers. Additionally, thecontainment valves 540 can be closed during thermocycling in order toprevent precipitation during the thermocycling process. A harvestingreagent flows from the harvesting port to the sample chamber (624) inorder to begin the process of harvesting the reaction products presentin the sample chambers. The harvesting port 136 is an example of a fluidinput port useful in the harvesting process. As illustrated in FIG. 4,the reaction products flow down through the sample input lines 516toward the sample input ports from which the samples were originallyprovides. Thus, in the illustrated method, removing the reactionproducts from the microfluidic device includes flowing the reactionproducts through at least a portion of the sample input line that wasused to load the samples to the sample input port. Thus, the reactionproduct are removed from the microfluidic device (626) and output to thesample input ports, for example, sample input ports 120 illustrated inFIG. 2.

Dilation pumping is used in the illustrated embodiment to remove thereaction products from the microfluidic device as discussed inadditional detail in relation to FIGS. 9A-D. Referring once again toFIG. 2, control ports 130 and 132 or pressure accumulators 150 and 152can be used to actuate the valves used in dilation pumping. Thus,embodiments provide valves for dilation pumping on the microfluidicdevice, which provides for removal of the reaction products from themicrofluidic device. This contrasts with conventional designs in whichsuch valves were not provided as part of the microfluidic device.

It should be appreciated that the specific steps illustrated in FIG. 7provide a particular method of operating a microfluidic device accordingto an embodiment of the present invention. Other sequences of steps mayalso be performed according to alternative embodiments. For example,alternative embodiments of the present invention may perform the stepsoutlined above in a different order. Moreover, the individual stepsillustrated in FIG. 7 may include multiple sub-steps that may beperformed in various sequences as appropriate to the individual step.Furthermore, additional steps may be added or removed depending on theparticular applications. One of ordinary skill in the art wouldrecognize many variations, modifications, and alternatives.

FIGS. 8A-8D are simplified schematic diagrams illustrating fluid flowthrough unit cells of a microfluidic device during operation accordingto an embodiment of the present invention. Referring to FIG. 8A, themicrofluidic device is illustrated during the sample and assay loadingprocess. Containment valves 540 are open, allowing fluid flow along thesample input lines 516 to the various sample chambers 510. The openstate of the containment valves also enables the assays to flow inthrough the assay input lines 518 to the various assay chambers 512.Using a single sample for each set of sample input lines (m of Msamples) enables loading of a single sample in each column of samplechambers. Additionally, using a single assay for each assay input line(n of N assays) enables loading of a single assay in each row of assaychambers. The closed state of the interface valves 530 prevent mixing ofthe samples and the assays.

FIG. 8B illustrates the microfluidic device during a sample and assaymixing process as well as a subsequent reaction process (e.g.,amplification). The containment valves 540 are closed, preventing fluidflow along the sample input lines 516. The closure of the containmentvalves thus isolates the sample chambers along a column from one another(with each column potentially containing a different sample). Theclosing of the containment valves 540 additionally isolates the assaychambers from other assay chambers in each row. After this chamberisolation is provided, the interface valves 530 are opened, enabling thesamples and assays to mix via free interface diffusion (FID) and formM×N pairwise combinations. Although the materials in the pairs ofsample/assay chambers are illustrated with the same shading in FIG. 8B,it will be appreciated that four different pairwise combinations areillustrated, one for each pair of sample/assay chambers. The multiplesteps involved in mixing the samples and assays, then performing PCRamplification, are represented by a single drawing in FIG. 8B althoughit will be apparent to one of skill in the art that numerous steps, forexample, multiple thermocycling steps, are involved in these processes.

FIG. 8C illustrates the microfluidic device during sample chamberisolation and initial loading of the harvesting reagent. The interfacevalves 530 are closed to maintain isolation between the sample chambersand the assay chambers. Thus, the reaction products present in thesample chambers are recovered while the reaction products in the assaychambers are not recovered. The containment valves 540 are opened toallow the harvesting reagent to flow into the sample input lines 516from the harvesting reagent input lines 430 illustrated in FIG. 5B. Theharvesting reagent flows through the sample input lines, and into thesample chambers. In the illustrated embodiment, the reaction productsare removed as the harvesting reagent flows through the sample inputlines and the sample chambers in response to a dilation pumping processdescribed in additional detail in relation to FIGS. 9A-9D. Asillustrated in FIG. 8C, the harvesting reagent has only reached themiddle region of the upper reaction chambers. As the dilation pumpingprocess continues, the harvesting reagent will be progressivelyintroduced into subsequent sample chambers, thereby displacing thereaction products. Eventually, the reaction products associated witheach sample will be recovered as a pooled fluid including the reactionproducts and the harvesting reagent in the sample input port from whichthe sample was originally dispensed.

It should be noted that the straight line representing the interfacebetween the harvesting reagent and the reaction products is shown merelyfor purposes of simplicity and it will be apparent to one of skill inthe art that in practice, a more complicated interface will be present.

FIG. 8D illustrates the microfluidic device during final loading of theharvesting reagent and recovery of the reaction products. As thedilation pumping process continues, harvesting reagent is introducedinto additional sample chambers progressively farther from theharvesting reagent input lines. The state of the recovery processillustrated in FIG. 8D shows that the reaction products have beenflushed from the sample chambers, which are now filled with harvestingreagent. Although only four sample chambers are illustrated in FIG. 8D,it will be appreciated that recovery of the reaction products from allthe sample chambers in the array is provided by the embodimentsdescribed herein.

FIGS. 9A-9D are simplified schematic diagrams illustrating fluid flowthrough a microfluidic device during operation according to anembodiment of the present invention. FIG. 9A illustrates a portion of amicrofluidic device according to an embodiment of the present inventionduring loading of samples and assays. The illustrated portion includes aharvesting reagent input line 810, vent and loading bowl portions 830,and isolation valve 840. As described more fully throughout the presentspecification, valves 820 and 822 are used to perform dilation pumpingof reaction products present in sample chambers 510. As illustrated inFIG. 9A, valve 820 is closed and valve 822 is open. Valves 820 and 822are typically “push-up” valves described elsewhere in the presentspecification.

Samples are loaded into sample chambers 510 and assays are loaded intoassay chambers 512 as described in relation to FIG. 6. Interface valves530 are closed, preventing mixing of the samples and assays. Vents andloading bowls are provided in some embodiments to allow for reductionsin effects related to depletion fronts. The inventors have observed thatin loading samples into microfluidic devices (e.g., through the verticalsample input lines illustrated in FIG. 9A), binding of a portion of thesample present at the leading edge of the flow path to the material ofthe microfluidic device will produce a depletion front in which one ormore components of the sample are depleted as a result of this bindingprocess. The provision of the vents and loading bowls 830 enables theuser to push the depletion front through the various sample chambers ofthe microfluidic device and store the depleted sample material in theloading bowls 830. Eventually, as the depleted sample material isflushed through the device into the loading bowls, the sample containedin the microfluidic device will be substantially undepleted.

Isolation valve 840 is open during the sample and assay loading processto enable the depletion front to flow into the loading bowls 830. Valve822 is open, allowing the samples to flow through the sample input linesto the various sample chambers. Since valve 820 is closed, samples arenot allowed to pass into the harvesting reagent input line 810. Itshould be noted that containment valves 540 are illustrated in theclosed state in FIG. 9A. Containment valves are open during sample andassay loading and then are closed as illustrated after sample and assayloading is complete. The containment valves isolate the various pairs ofreaction and assay chambers from other pairs containing the variouspairwise combinations.

In FIG. 9A, only a single column of the microfluidic device isillustrated for purposes of clarity. It is understood that additionalcolumns are provided by the microfluidic devices as illustrated, forexample, in FIG. 6. Moreover, much of the column is not illustrated forthe purposes of clarity. The two sets of sample/assay chambersillustrated are those at the top and bottom of FIG. 4A, respectively.The set adjacent valve 820 is the topmost set and the set adjacent valve822 is the bottommost set. Thus, these diagrams are merelyrepresentative and not intended to limit the scope of the presentinvention.

FIG. 9B illustrates mixing of the samples and assays and a subsequentreaction (e.g., amplification) process. In order to mix the samples andreagents, interface valves 530 are placed in the open position as shown.Closure of containment valves 540 seals the reaction products in thesample and assay chambers along with the connecting fluid lines. Asillustrated in FIG. 9B, isolation valve 840 is closed, preventing fluidflow between the sample input lines and the loading bowls 830. Actuationof valve 840 to place it in the open or closed position is performedusing a pressure accumulator (not shown) in some embodiments and usingother actuation techniques in other embodiments, for example,mechanical, electrostatic, electromechanical, thermodynamic,piezoelectric, or the like. Such additional techniques may also beapplicable to other valves described herein. One of ordinary skill inthe art would recognize many variations, modifications, andalternatives.

Although FIG. 9B illustrates mixing and reaction using a single image,one of skill in the art will appreciate that multiple thermal cycles maybe used to amplify DNA using the PCR process. Thus, this simple figureis intended to show mixing and subsequent reactions that can occur inthe microfluidic device.

FIG. 9C illustrates a portion of the microfluidic device in a firststage of a reaction product harvesting process. A harvesting reagentflows from a harvesting port as illustrated in FIG. 5A throughharvesting reagent input line 810 toward the sample chambers. As shownin FIG. 9A, valve 820 is open, allowing the harvesting reagent to flowinto the topmost sample chamber. Closure of the interface valves 530prevents the harvesting reagent from flowing into the assay chambers,which also contain reaction products. The extent to which the harvestingreagent initially fills the sample input lines and sample input chambersis limited by the closure of valve 822. As illustrated, the harvestingreagent has only partially filled a portion of the first sample chamber.The illustration of a side of the sample chamber being filled withharvesting reagent is merely provided by way of example, since the flowof the harvesting reagent into the sample chamber is actually throughvias extending from the plane of the figure. Closure of isolation valve840 prevents harvesting reagent from flowing into the loading bowls,although other embodiments may enable such a flow if desired.

Fluid pressure resulting from the flow of the harvesting reagent intothe array portion of the microfluidic device results in expansion of thesample input lines and sample chambers above the valve 822. The pumpcycle is initiated by this pressurization of the sample chambers. Asdescribed below, closing of valve 820 and opening of valve 822 willenable the pressurized harvesting reagent and reaction products to berecovered from the microfluidic device as it flows through themicrofluidic device.

FIG. 9D illustrates a portion of the microfluidic device in a secondstage of the reaction product harvesting process. Although a secondstage is illustrated, this is not intended to imply that the secondstage immediately follows the first stage. As described below, thesecond stage is typically separated from the first stage by one or moreintermediate stages of dilation pumping.

Dilation pumping (also know as volumetric capacitive pumping) is amethod of operating a properly configured integrated fluidic circuit(microfluidic device) to obtain precise, low rate, low volume pumpingthrough all configured elements of the microfluidic device. Dilationpumping is unique to microfluidic circuits that utilize channels thathave one or more channel walls formed from an elastomeric material. Asan example, the flow of the harvesting reagent through the sample inputlines and sample chambers is considered volumetric capacitive pumping.Pumping proceeds by the closure of valves 822 and the opening of valves820. As discussed above, harvesting reagent ports (not illustrated) arepressurized to introduce the harvesting reagent into the topmost sampleinput lines and sample chambers, which can be considered as a channel.The pressurization of microfluidic channels with at least one channelwall formed from an elastomeric material results in expansion of theelastomeric wall(s) outward from the channel with a resulting increasein channel volume that is proportional to the fluidic pressure (orgaseous pressure in alternate embodiments) within the channel, theelastic properties of the elastomeric channel wall material such asYoung's modulus, and the length and cross sectional area of the channel.The sample input lines and sample chambers are allowed to pressurize andthen valves 820 is closed as illustrated in FIG. 9D. Following closureof valves 820, valves 822 are opened. The pumped volume through thesample input lines and the sample chambers is equal to the expandedvolume of the channel when under pressure minus the native volume of thechannel when pressure is released and the expanded elastomeric channelwall(s) is allow to relax. Dilation pumping is continued throughrepetitive cycles of closing 822, opening 820, pressurizing the sampleinput lines and sample chambers, closing 820, and opening 822. In thismanner, continuous or discontinuous low volume pumping may beaccomplished at precisely controlled flow rates.

Thus embodiments provide a method of dilation pumping that includesclosing a first valve disposed between the sample chamber and the sampleinput port (i.e., valve 822), opening a second valve disposed betweenthe harvesting port and the sample chamber (i.e., valve 820), closingthe second valve, opening the first valve, and repeating these steps apredetermined number of times. Between the steps of opening the secondvalve and closing the second valve, the harvesting reagent flows intothe sample input lines and sample chambers, pressurizing the channel asdescribed above. After the dilation pumping process is complete,harvesting reagent substantially fills the sample input lines and samplechambers (e.g., recovery rates >95%), thereby pooling the reactionproducts associated with a given sample in the sample input port fromwhich the given sample was initially dispensed.

Dilation pumping provides benefits not typically available usingconventional techniques. For example, dilation pumping enables for aslow removal of the reaction products from the microfluidic device. Inan exemplary embodiment, the reaction products are recovered at a fluidflow rate of less than 100 μl per hour. In this example, for 48 reactionproducts distributed among the reaction chambers in each column, with avolume of each reaction product of about 1.5 μl, removal of the reactionproducts in a period of about 30 minutes, will result in a fluid flowrate of 72 μl/hour. (i.e., 48*1.5/0.5 hour). In other embodiments, theremoval rate of the reaction products is performed at a rate of lessthan 90 μl/hr, 80 μl/hr, 70 μl/hr, 60 μl/hr, 50 μl/hr, 40 μl/hr, 30μl/hr, 20 μl/hr, 10 μl/hr, 9 μl/hr, less than 8 μl/hr, less than 7μl/hr, less than 6 μl/hr, less than 5 μl/hr, less than 4 μl/hr, lessthan 3 μl/hr, less than 2 μl/hr, less than 1 μl/hr, or less than 0.5μl/hr.

Dilation pumping results in clearing of substantially a high percentageand potentially all the reaction products present in the microfluidicdevice. Some embodiments remove more than 75% of the reaction productspresent in the reaction chambers (e.g., sample chambers) of themicrofluidic device. As an example, some embodiments remove more than80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% of the reaction productspresent in the reaction chambers.

In some embodiments, a harvesting valve is provided on the microfluidicdevice to obstruct the flow of harvesting reagent through the device.Application of a pressure source to a harvesting input port results inflow of harvesting fluid (e.g., a harvesting liquid) through harvestreagent input lines up to the harvesting valve. The permeability of thematerials utilized to fabricate the microfluidic device enables such aharvesting fluid to fill the harvest reagent input lines, typicallyexpelling air initially present in such lines. The presence of theharvesting valve will obstruct the flow of the harvest reagent at thelocation of the harvesting valve. Actuation (i.e., opening) of theharvesting valve will result in the harvesting fluid flowing through theharvest reagent input lines downstream of the harvesting valve. In otherembodiments, a harvesting valve is replaced with one or more othersuitable valves as appropriate to the particular application. Forexample, in the embodiment illustrated in FIGS. 9A-9D, valve 820 servesto prevent flow of harvesting reagent until the dilation pumping processis initiated.

Fabrication methods using elastomeric materials and methods for designof devices and their components have been described in detail in thescientific and patent literature. See, e.g., Unger et al. (2000) Science288:113-116; U.S. Pat. Nos. U.S. Pat. No. 6,960,437 (Nucleic acidamplification utilizing microfluidic devices); U.S. Pat. No. 6,899,137(Microfabricated elastomeric valve and pump systems); U.S. Pat. No.6,767,706 (Integrated active flux microfluidic devices and methods);U.S. Pat. No. 6,752,922 (Microfluidic chromatography); U.S. Pat. No.6,408,878 (Microfabricated elastomeric valve and pump systems); U.S.Pat. No. 6,645,432 (Microfluidic systems including three-dimensionallyarrayed channel networks); U.S. Patent Application Publication Nos.2004/0115838; 2005/0072946; 2005/0000900; 2002/0127736; 2002/0109114;2004/0115838; 2003/0138829; 2002/0164816; 2002/0127736; and2002/0109114; PCT Publication Nos. WO 2005/084191; WO 05/030822A2; andWO 01/01025; Quake & Scherer, 2000, “From micro to nanofabrication withsoft materials” Science 290: 1536-40; Unger et al., 2000, “Monolithicmicrofabricated valves and pumps by multilayer soft lithography” Science288:113-116; Thorsen et al., 2002, “Microfluidic large-scaleintegration” Science 298:580-584; Chou et al., 2000, “MicrofabricatedRotary Pump” Biomedical Microdevices 3:323-330; Liu et al., 2003,“Solving the “world-to-chip” interface problem with a microfluidicmatrix” Analytical Chemistry 75, 4718-23, Hong et al, 2004, “Ananoliter-scale nucleic acid processor with parallel architecture”Nature Biotechnology 22:435-39.

According to certain embodiments describer herein, the detection and/orquantification of one or more target nucleic acids from one or moresamples may generally be carried out on a microfluidic device byobtaining a sample, optionally pre-amplifying the sample, anddistributing the optionally pre-amplified sample, or aliquots thereof,into reaction chambers of a microfluidic device containing theappropriate buffers, primers, optional probe(s), and enzyme(s),subjecting these mixtures to amplification, and querying the aliquotsfor the presence of amplified target nucleic acids. The sample aliquotsmay have a volume of less than 1 picoliter or, in various embodiments,in the range of about 1 picoliter to about 500 nanoliters, in a range ofabout 2 picoliters to about 50 picoliters, in a range of about 5picoliters to about 25 picoliters, in the range of about 100 picolitersto about 20 nanoliters, in the range of about 1 nanoliter to about 20nanoliters, and in the range of about 5 nanoliters to about 15nanoliters. In many embodiments, sample aliquots account for themajority of the volume of the amplification mixtures. Thus,amplification mixtures can have a volume of less than 1 picoliter or, invarious embodiments about 2, about 5 about 7, about 10, about 15, about20, about 25, about 50, about 100, about 250, about 500, and about 750picoliters; or about 1, about 2, about 5, about 7, about 15, about 20,about 25, about 50, about 250, and about 500 nanoliters. Theamplification mixtures can also have a volume within any range boundedby any of these values (e.g., about 2 picoliters to about 50picoliters).

In certain embodiments, multiplex detection is carried out in individualamplification mixture, e.g., in individual reaction chambers of amicrofluidic device, which can be used to further increase the number ofsamples and/or targets that can be analyzed in a single assay or tocarry out comparative methods, such as comparative genomic hybridization(CGH). In various embodiments, up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 50,100, 500, 1000, 5000, 10000 or more amplification reactions are carriedout in each individual reaction chamber.

In specific embodiments, the assay usually has a dynamic range of atleast 3 orders of magnitude, more often at least 4, at least 5, at least6, at least 7, or at least 8 orders of magnitude.

Quantitative Real-Time PCR and Other Detection and QuantificationMethods

Any method of detection and/or quantification of nucleic acids can beused in the invention to detect amplification products. In oneembodiment, PCR (polymerase chain reaction) is used to amplify and/orquantify target nucleic acids. In other embodiments, other amplificationsystems or detection systems are used, including, e.g., systemsdescribed in U.S. Pat. No. 7,118,910 (which is incorporated herein byreference in its entirety for its description of amplification/detectionsystems) and Invader assays; PE BioSystems). In particular embodiments,real-time quantification methods are used. For example, “quantitativereal-time PCR” methods can be used to determine the quantity of a targetnucleic acid present in a sample by measuring the amount ofamplification product formed during the amplification process itself.

Fluorogenic nuclease assays are one specific example of a real-timequantification method that can be used successfully in the methodsdescribed herein. This method of monitoring the formation ofamplification product involves the continuous measurement of PCR productaccumulation using a dual-labeled fluorogenic oligonucleotide probe—anapproach frequently referred to in the literature as the “TaqMan®method.” See U.S. Pat. No. 5,723,591; Heid et al., 1996, Real-timequantitative PCR Genome Res. 6:986-94, each incorporated herein byreference in their entireties for their descriptions of fluorogenicnuclease assays. It will be appreciated that while “TaqMan® probes” arethe most widely used for qPCR, the invention is not limited to use ofthese probes; any suitable probe can be used.

Other detection/quantification methods that can be employed in thepresent invention include FRET and template extension reactions,molecular beacon detection, Scorpion detection, Invader detection, andpadlock probe detection.

FRET and template extension reactions utilize a primer labeled with onemember of a donor/acceptor pair and a nucleotide labeled with the othermember of the donor/acceptor pair. Prior to incorporation of the labelednucleotide into the primer during a template-dependent extensionreaction, the donor and acceptor are spaced far enough apart that energytransfer cannot occur. However, if the labeled nucleotide isincorporated into the primer and the spacing is sufficiently close, thenenergy transfer occurs and can be detected. These methods areparticularly useful in conducting single base pair extension reactionsin the detection of single nucleotide polymorphisms and are described inU.S. Pat. No. 5,945,283 and PCT Publication WO 97/22719.

With molecular beacons, a change in conformation of the probe as ithybridizes to a complementary region of the amplified product results inthe formation of a detectable signal. The probe itself includes twosections: one section at the 5′ end and the other section at the 3′ end.These sections flank the section of the probe that anneals to the probebinding site and are complementary to one another. One end section istypically attached to a reporter dye and the other end section isusually attached to a quencher dye. In solution, the two end sectionscan hybridize with each other to form a hairpin loop. In thisconformation, the reporter and quencher dye are in sufficiently closeproximity that fluorescence from the reporter dye is effectivelyquenched by the quencher dye. Hybridized probe, in contrast, results ina linearized conformation in which the extent of quenching is decreased.Thus, by monitoring emission changes for the two dyes, it is possible toindirectly monitor the formation of amplification product. Probes ofthis type and methods of their use are described further, for example,by Piatek et al., 1998, Nat. Biotechnol. 16:359-63; Tyagi, and Kramer,1996, Nat. Biotechnology 14:303-308; and Tyagi, et al., 1998, Nat.Biotechnol. 16:49-53 (1998).

The Scorpion detection method is described, for example, by Thelwell etal. 2000, Nucleic Acids Research, 28:3752-3761 and Solinas et al., 2001,“Duplex Scorpion primers in SNP analysis and FRET applications” NucleicAcids Research 29:20. Scorpion primers are fluorogenic PCR primers witha probe element attached at the 5′-end via a PCR stopper. They are usedin real-time amplicon-specific detection of PCR products in homogeneoussolution. Two different formats are possible, the “stem-loop” format andthe “duplex” format. In both cases the probing mechanism isintramolecular. The basic elements of Scorpions in all formats are: (i)a PCR primer; (ii) a PCR stopper to prevent PCR read-through of theprobe element; (iii) a specific probe sequence; and (iv) a fluorescencedetection system containing at least one fluorophore and quencher. AfterPCR extension of the Scorpion primer, the resultant amplicon contains asequence that is complementary to the probe, which is renderedsingle-stranded during the denaturation stage of each PCR cycle. Oncooling, the probe is free to bind to this complementary sequence,producing an increase in fluorescence, as the quencher is no longer inthe vicinity of the fluorophore. The PCR stopper prevents undesirableread-through of the probe by Taq DNA polymerase.

Invader assays (Third Wave Technologies, Madison, Wis.) are usedparticularly for SNP genotyping and utilize an oligonucleotide,designated the signal probe, that is complementary to the target nucleicacid (DNA or RNA) or polymorphism site. A second oligonucleotide,designated the Invader Oligo, contains the same 5′ nucleotide sequence,but the 3′ nucleotide sequence contains a nucleotide polymorphism. TheInvader Oligo interferes with the binding of the signal probe to thetarget nucleic acid such that the 5′ end of the signal probe forms a“flap” at the nucleotide containing the polymorphism. This complex isrecognized by a structure specific endonuclease, called the Cleavaseenzyme. Cleavase cleaves the 5′ flap of the nucleotides. The releasedflap binds with a third probe bearing FRET labels, thereby forminganother duplex structure recognized by the Cleavase enzyme. This time,the Cleavase enzyme cleaves a fluorophore away from a quencher andproduces a fluorescent signal. For SNP genotyping, the signal probe willbe designed to hybridize with either the reference (wild type) allele orthe variant (mutant) allele. Unlike PCR, there is a linear amplificationof signal with no amplification of the nucleic acid. Further detailssufficient to guide one of ordinary skill in the art are provided by,for example, Neri, B. P., et al., Advances in Nucleic Acid and ProteinAnalysis 3826:117-125, 2000) and U.S. Pat. No. 6,706,471.

Padlock probes (PLPs) are long (e.g., about 100 bases) linearoligonucleotides. The sequences at the 3′ and 5′ ends of the probe arecomplementary to adjacent sequences in the target nucleic acid. In thecentral, noncomplementary region of the PLP there is a “tag” sequencethat can be used to identify the specific PLP. The tag sequence isflanked by universal priming sites, which allow PCR amplification of thetag. Upon hybridization to the target, the two ends of the PLPoligonucleotide are brought into close proximity and can be joined byenzymatic ligation. The resulting product is a circular probe moleculecatenated to the target DNA strand. Any unligated probes (i.e., probesthat did not hybridize to a target) are removed by the action of anexonuclease. Hybridization and ligation of a PLP requires that both endsegments recognize the target sequence. In this manner, PLPs provideextremely specific target recognition.

The tag regions of circularized PLPs can then be amplified and resultingamplicons detected. For example, TaqMan® real-time PCR can be carriedout to detect and quantify the amplicon. The presence and amount ofamplicon can be correlated with the presence and quantity of targetsequence in the sample. For descriptions of PLPs see, e.g., Landegren etal., 2003, Padlock and proximity probes for in situ and array-basedanalyses: tools for the post-genomic era, Comparative and FunctionalGenomics 4:525-30; Nilsson et al., 2006, Analyzing genes using closingand replicating circles Trends Biotechnol. 24:83-8; Nilsson et al.,1994, Padlock probes: circularizing oligonucleotides for localized DNAdetection, Science 265:2085-8.

In particular embodiments, fluorophores that can be used as detectablelabels for probes include, but are not limited to, rhodamine, cyanine 3(Cy 3), cyanine 5 (Cy 5), fluorescein, Vic™, Liz™, Tamra™, 5-Fam™,6-Fam™, and Texas Red (Molecular Probes). (Vic™, Liz™, Tamra™, 5-Fam™,6-Fam™ are all available from Applied Biosystems, Foster City, Calif.).

Devices have been developed that can perform a thermal cycling reactionwith compositions containing a fluorescent indicator, emit a light beamof a specified wavelength, read the intensity of the fluorescent dye,and display the intensity of fluorescence after each cycle. Devicescomprising a thermal cycler, light beam emitter, and a fluorescentsignal detector, have been described, e.g., in U.S. Pat. Nos. 5,928,907;6,015,674; and 6,174,670.

In some embodiments, each of these functions can be performed byseparate devices. For example, if one employs a Q-beta replicasereaction for amplification, the reaction may not take place in a thermalcycler, but could include a light beam emitted at a specific wavelength,detection of the fluorescent signal, and calculation and display of theamount of amplification product.

In particular embodiments, combined thermal cycling and fluorescencedetecting devices can be used for precise quantification of targetnucleic acids. In some embodiments, fluorescent signals can be detectedand displayed during and/or after one or more thermal cycles, thuspermitting monitoring of amplification products as the reactions occurin “real-time.” In certain embodiments, one can use the amount ofamplification product and number of amplification cycles to calculatehow much of the target nucleic acid sequence was in the sample prior toamplification.

According to some embodiments, one can simply monitor the amount ofamplification product after a predetermined number of cycles sufficientto indicate the presence of the target nucleic acid sequence in thesample. One skilled in the art can easily determine, for any givensample type, primer sequence, and reaction condition, how many cyclesare sufficient to determine the presence of a given target nucleic acid.

According to certain embodiments, one can employ an internal standard toquantify the amplification product indicated by the fluorescent signal.See, e.g., U.S. Pat. No. 5,736,333.

In various embodiments, employing preamplification, the number ofpreamplification cycles is sufficient to add one or more nucleotide tagsto the target nucleotide sequences, so that the relative copy numbers ofthe tagged target nucleotide sequences is substantially representativeof the relative copy numbers of the target nucleic acids in the sample.For example, preamplification can be carried out for 2-20 cycles tointroduce the sample-specific or set-specific nucleotide tags. In otherembodiments, detection is carried out at the end of exponentialamplification, i.e., during the “plateau” phase, or endpoint PCR iscarried out. In this instance, preamplification will normalize ampliconcopy number across targets and across samples. In various embodiments,preamplification and/or amplification can be carried out for about: 2,4, 10, 15, 20, 25, 30, 35, or 40 cycles or for a number of cyclesfalling within any range bounded by any of these values.

Labeling Strategies

Any suitable labeling strategy can be employed in the methods of theinvention. Where the assay mixture is aliquoted, and each aliquot isanalyzed for presence of a single amplification product, a universaldetection probe can be employed in the amplification mixture. Inparticular embodiments, real-time PCR detection can be carried out usinga universal qPCR probe. Suitable universal qPCR probes includedouble-stranded DNA dyes, such as SYBR Green, Pico Green (MolecularProbes, Inc., Eugene, Oreg.), Eva Green (Biotinum), ethidium bromide,and the like (see Zhu et al., 1994, Anal. Chem. 66:1941-48). Suitableuniversal qPCR probes also include sequence-specific probes that bind toa nucleotide sequence present in all amplification products. Bindingsites for such probes can be conveniently introduced into the taggedtarget nucleic acids during amplification.

Alternatively, one or more target-specific qPCR probes (i.e., specificfor a target nucleotide sequence to be detected) is employed in theamplification mixtures to detect amplification products. Target-specificprobes could be useful, e.g., when only a few target nucleic acids areto be detected in a large number of samples. For example, if only threetargets were to be detected, a target-specific probe with a differentfluorescent label for each target could be employed. By judicious choiceof labels, analyses can be conducted in which the different labels areexcited and/or detected at different wavelengths in a single reaction.See, e.g., Fluorescence Spectroscopy (Pesce et al., Eds.) Marcel Dekker,New York, (1971); White et al., Fluorescence Analysis: A PracticalApproach, Marcel Dekker, New York, (1970); Berlman, Handbook ofFluorescence Spectra of Aromatic Molecules, 2nd ed., Academic Press, NewYork, (1971); Griffiths, Colour and Constitution of Organic Molecules,Academic Press, New York, (1976); Indicators (Bishop, Ed.). PergamonPress, Oxford, 19723; and Haugland, Handbook of Fluorescent Probes andResearch Chemicals, Molecular Probes, Eugene (1992).

Removal of Undesired Reaction Components

It will be appreciated that reactions involving complex mixtures ofnucleic acids in which a number of reactive steps are employed canresult in a variety of unincorporated reaction components, and thatremoval of such unincorporated reaction components, or reduction oftheir concentration, by any of a variety of clean-up procedures canimprove the efficiency and specificity of subsequently occurringreactions. For example, it may be desirable, in some embodiments, toremove, or reduce the concentration of preamplification primers prior tocarrying out the amplification steps described herein.

In certain embodiments, the concentration of undesired components can bereduced by simple dilution. For example, preamplified samples can bediluted about 2-, 5-, 10-, 50-, 100-, 500-, 1000-fold prior toamplification to improve the specificity of the subsequent amplificationstep.

In some embodiments, undesired components can be removed by a variety ofenzymatic means. Alternatively, or in addition to the above-describedmethods, undesired components can be removed by purification. Forexample, a purification tag can be incorporated into any of theabove-described primers (e.g., into the barcode nucleotide sequence) tofacilitate purification of the tagged target nucleotides.

In particular embodiments, clean-up includes selective immobilization ofthe desired nucleic acids. For example, desired nucleic acids can bepreferentially immobilized on a solid support. In an illustrativeembodiment, an affinity moiety, such as biotin (e.g., photo-biotin), isattached to desired nucleic acid, and the resulting biotin-labelednucleic acids immobilized on a solid support comprising an affinitymoiety-binder such as streptavidin. Immobilized nucleic acids can bequeried with probes, and non-hybridized and/or non-ligated probesremoved by washing (See, e.g., Published P.C.T. Application WO 03/006677and U.S. Ser. No. 09/931,285.) Alternatively, immobilized nucleic acidscan be washed to remove other components and then released from thesolid support for further analysis. This approach can be used, forexample, in recovering target amplicons from amplification mixturesafter the addition of primer binding sites for DNA sequencing. Inparticular embodiments, an affinity moiety, such as biotin, can beattached to an amplification primer such that amplification produces anaffinity moiety-labeled (e.g., biotin-labeled) amplicon. Thus, forexample, where three primers are employed to add barcode and nucleotidetag elements to a target nucleotide sequence, as described above, atleast one of the barcode or reverse primers can include an affinitymoiety. Where four primers (two inner primers and two outer primers) areemployed to add desired element to a target nucleotide sequence, atleast one of the outer primers can include an affinity moiety.

Data Output and Analysis

In certain embodiments, when the methods of the invention are carriedout on a matrix-type microfluidic device, the data can be output as aheat matrix (also termed “heat map”). In the heat matrix, each square,representing a reaction chamber on the DA matrix, has been assigned acolor value which can be shown in gray scale, but is more typicallyshown in color. In gray scale, black squares indicate that noamplification product was detected, whereas white squares indicate thehighest level of amplification produce, with shades of gray indicatinglevels of amplification product in between. In a further aspect, asoftware program may be used to compile the data generated in the heatmatrix into a more reader-friendly format.

Applications

The methods of the invention are applicable to any technique aimed atdetecting the presence or amount of one or more target nucleic acids ina nucleic acid sample. Thus, for example, these methods are applicableto identifying the presence of particular polymorphisms (such as SNPs),alleles, or haplotypes, or chromosomal abnormalities, such asamplifications, deletions, or aneuploidy. The methods may be employed ingenotyping, which can be carried out in a number of contexts, includingdiagnosis of genetic diseases or disorders, pharmacogenomics(personalized medicine), quality control in agriculture (e.g., for seedsor livestock), the study and management of populations of plants oranimals (e.g., in aquaculture or fisheries management or in thedetermination of population diversity), or paternity or forensicidentifications. The methods of the invention can be applied in theidentification of sequences indicative of particular conditions ororganisms in biological or environmental samples. For example, themethods can be used in assays to identify pathogens, such as viruses,bacteria, and fungi). The methods can also be used in studies aimed atcharacterizing environments or microenvironments, e.g., characterizingthe microbial species in the human gut.

These methods can also be employed in determinations DNA or RNA copynumber. Determinations of aberrant DNA copy number in genomic DNA isuseful, for example, in the diagnosis and/or prognosis of geneticdefects and diseases, such as cancer. Determination of RNA “copynumber,” i.e., expression level is useful for expression monitoring ofgenes of interest, e.g., in different individuals, tissues, or cellsunder different conditions (e.g., different external stimuli or diseasestates) and/or at different developmental stages.

In addition, the methods can be employed to prepare nucleic acid samplesfor further analysis, such as, e.g., DNA sequencing.

Finally, nucleic acid samples can be tagged as a first step, priorsubsequent analysis, to reduce the risk that mislabeling orcross-contamination of samples will compromise the results. For example,any physician's office, laboratory, or hospital could tag samplesimmediately after collection, and the tags could be confirmed at thetime of analysis. Similarly, samples containing nucleic acids collectedat a crime scene could be tagged as soon as practicable, to ensure thatthe samples could not be mislabeled or tampered with. Detection of thetag upon each transfer of the sample from one party to another could beused to establish chain of custody of the sample.

Kits

Kits according to the invention include one or more reagents useful forpracticing one or more assay methods of the invention. A kit generallyincludes a package with one or more containers holding the reagent(s)(e.g., primers and/or probe(s)), as one or more separate compositionsor, optionally, as admixture where the compatibility of the reagentswill allow. The kit can also include other material(s) that may bedesirable from a user standpoint, such as a buffer(s), a diluent(s), astandard(s), and/or any other material useful in sample processing,washing, or conducting any other step of the assay.

Kits according to the invention generally include instructions forcarrying out one or more of the methods of the invention. Instructionsincluded in kits of the invention can be affixed to packaging materialor can be included as a package insert. While the instructions aretypically written or printed materials they are not limited to such. Anymedium capable of storing such instructions and communicating them to anend user is contemplated by this invention. Such media include, but arenot limited to, electronic storage media (e.g., magnetic discs, tapes,cartridges, chips), optical media (e.g., CD ROM), RF tags, and the like.As used herein, the term “instructions” can include the address of aninternet site that provides the instructions.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

In addition, all other publications, patents, and patent applicationscited herein are hereby incorporated by reference in their entirety forall purposes.

EXAMPLES Example 1 Multi-Primer Amplification Method for Barcoding ofTarget Nucleic Acids in Preparation for DNA Sequencing

Genomic DNA samples (BioChain, USA) at 100 and 0 ng/ml (negative control[“NTC”]) were amplified for 25 cycles 7900HT Fast Real-Time PCR System(Applied Biosystems, USA) with the following primer pairs at 200 nM perprimer: 1) 454 tails; 2) A5 specific primers; and 3) the three primersshown in FIG. 10. PCR was performed in 15 μl reaction volumes containing7.5 μl of FastStart TaqMan® Probe Master (Roche Diagnostics, USA), 0.75μl of DA sample loading reagent (Fluidigm Corp, USA) and 6.75 μl ofsample. Thermal cycling condition included an initial hot start at 50°C. for 2 minutes and at 94° C. for 10 minutes, followed by 25 cycles at94° C. for 15 s, 70° C. for 5 s, 60° C. for 30 s and 72° C. for 90 s.The resulting amplification products were run on an electrophoresis gel(Invitrogen, USA) using 8 μl of the reaction mixture per lane followingthe manufacturer instruction. See FIG. 11, which shows that the 3-primermethod produced an amplicon of the correct size. The amplicons generatedfrom PCR amplification were purified using Ampure Beads® and thenre-amplified on a PCR plate with 454 tail primers, followed by Sangersequencing with either 454 tail primers, which showed that the 3-primermethod generated an amplicon having the correct sequence.

Example 2 Multi-Primer Amplification Method for Quantifying TargetNucleic Acids in Preparation for DNA Sequencing

Primers for preparing genomic DNA for sequencing using various DNAconventional DNA sequencing methods are shown below.

ShotGun Forward: (SEQ ID NO: 1) 5′-CCATCTCATCCCTGCGTGTC-3′ShotGun Reverse: (SEQ ID NO: 2) 5′-CCTATCCCCTGTGTGCCTTG-3′ShotGun UPR Forward: (SEQ ID NO: 3) 5′-GGCGGCGACCATCTCATCCCTGCGTGTC-3′MID Forward: (SEQ ID NO: 4) 5′-GCCTCCCTCGCGCCATCAG-3′ MID Reverse:(SEQ ID NO: 5) 5′-GCCTTGCCAGCCCGCTCAG-3′ MID UPR Forward: (SEQ ID NO: 6)5′-GGCGGCGAGCCTCCCTCGCGCCATCAG-3′ Solexa Forward: (SEQ ID NO: 7)5′-ACACTCTTTCCCTACACGA-3′ Solexa Reverse: (SEQ ID NO: 8)5′-CAAGCAGAAGACGGCATA-3′ Solexa UPR Forward: (SEQ ID NO: 9)5′-GGCGGCGAACACTCTTTCCCTACACGA-3′ Solid Forward: (SEQ ID NO: 10)5′-CCACTACGCCTCCGCTTTCCTCTCTATG-3′ Solid Reverse: (SEQ ID NO: 11)5′-CTGCCCCGGGTTCCTCATTCT-3′ Solid UPR Forward:  (SEQ ID NO: 12)5′-GGCGGCGACCACTACGCCTCCGCTTTCCTCTCTATG-3′ 454 Titanium Forward:(SEQ ID NO: 13) 5′-CCATCTCATCCCTGCGTG-3′ 454 Titanium Reverse:(SEQ ID NO: 14) 5′-CCTATCCCCTGTGTGCCTTG-3′ 454 Titanium UPR Forward: (SEQ ID NO: 15) 5′-GGCGGCGACCATCTCATCCCTGCGTG-3′ Solexa smRNA Forward:(SEQ ID NO: 16) 5′-TAATGATACGGCGACCACC-3′ Solexa smRNA Reverse:(SEQ ID NO: 17) 5′-ACAAGCAGAAGACGGCATAC-3′ Solexa smRNA UPL Forward:(SEQ ID NO: 18) 5′-GGCGGCGATAATGATACGGCGACCAC-3′

The properties of these primers is shown in Table 1 below.

TABLE 1 Primer Length (nt) CG % Tm (° C.) Primer-Dimer 454- ShotGunForward: 20 60 68.4 No self/cross-dimer, 1.5° C. diff standard in Tm(ShotGun) ShotGun Reverse: 20 60 66.9 ShotGun UPR 28 67.8 84.8 Forward:454-MID MID Forward: 19 73.6 74.9 4-bases of self-dimer(F.UPL) &cross-dimer(F./UPL, R/UPL) MID Reverse: 19 73.6 74.9 High GC MID UPRForward: 27 77.7 88.5 Solexa Solexa Forward: 19 47.3 57.8 No dimer, 2.1°C. diff in Tm Solexa Reverse: 18 50 60.6 Low GC Solexa UPR 27 59.2 78.4Forward: Solid Solid Forward: 28 57.1 74.7 Strong self-dimer & cross-dimer Solid Reverse: 21 61.9 72.5 variety of GC & Tm Solid UPR Forward:36 63.8 85.6

The reaction mixture used for amplification of genomic DNA toincorporate primer sequences is given below in Table 2.

TABLE 2 Add V μl of TE into dry 100 uM stock probe tube solution V =“Total nmol” value of 10X Fluidigm the dry probe * 10 Assay 100 μMolForward: 4 2000 nM UPR Forward: 4 2000 nM Reverse: 8 4000 nM TE: 184Total: 200

Example 3 Additional Illustrative Primers for Barcoding of TargetNucleic Acids in Preparation for 454 DNA Sequencing

Tables 3 and 4 below show additional illustrative primers for barcodingof target nucleic acids in preparation for 454 DNA sequencing. “454F”refers to a 454 forward primer binding site; “454R” refers to 454reverse primer binding site. “BC” refers to a nucleotide barcode. “TAG”refers to a nucleotide tag. “P53” refers to a target-specific primersequence.

TABLE 3 Sequence Name Sequence SEQ ID 454F-BC1-TAG8GCCTCCCTCGCGCCATCAGGCATGCACACTGACGACA (SEQ ID NO: 19) TGGTTCTACA454F-BC2-TAG8 GCCTCCCTCGCGCCATCAGCGTACGACACTGACGACA (SEQ ID NO: 20)TGGTTCTACA 454F-BC3-TAG8 GCCTCCCTCGCGCCATCAGGTCAGCACACTGACGACA(SEQ ID NO: 21) TGGTTCTACA 454F-BC4-TAG8GCCTCCCTCGCGCCATCAGAGCTGCACACTGACGACA (SEQ ID NO: 22) TGGTTCTACA454F-BC5-TAG8 GCCTCCCTCGCGCCATCAGTGCATCACACTGACGACA (SEQ ID NO: 23)TGGTTCTACA 454F-BC6-TAG8 GCCTCCCTCGCGCCATCAGCTGATGACACTGACGACA(SEQ ID NO: 24) TGGTTCTACA 454F-BC7-TAG8GCCTCCCTCGCGCCATCAGGTAGTCACACTGACGACA (SEQ ID NO: 25) TGGTTCTACA454F-BC8-TAG8 GCCTCCCTCGCGCCATCAGGTCGATACACTGACGACA (SEQ ID NO: 26)TGGTTCTACA 454F-BC9-TAG8 GCCTCCCTCGCGCCATCAGGATACGACACTGACGACA(SEQ ID NO: 27) TGGTTCTACA 454F-BC10-TAG8GCCTCCCTCGCGCCATCAGTGATGCACACTGACGACA (SEQ ID NO: 28) TGGTTCTACA454F-BC11-TAG8 GCCTCCCTCGCGCCATCAGAGCTGAACACTGACGACA (SEQ ID NO: 29)TGGTTCTACA 454F-BC12-TAG8 GCCTCCCTCGCGCCATCAGACTGTAACACTGACGACA(SEQ ID NO: 30) TGGTTCTACA 454F-BC13-TAG8GCCTCCCTCGCGCCATCAGTGCATGACACTGACGACA (SEQ ID NO: 31) TGGTTCTACA454F-BC14-TAG8 GCCTCCCTCGCGCCATCAGAGTCTAACACTGACGACA (SEQ ID NO: 32)TGGTTCTACA 454F-BC15-TAG8 GCCTCCCTCGCGCCATCAGTGTCTGACACTGACGACA(SEQ ID NO: 33) TGGTTCTACA 454F-BC16-TAG8GCCTCCCTCGCGCCATCAGGCTAGCACACTGACGACA (SEQ ID NO: 34) TGGTTCTACA454F-BC17-TAG8 GCCTCCCTCGCGCCATCAGGATAGCACACTGACGACA (SEQ ID NO: 35)TGGTTCTACA 454F-BC18-TAG8 GCCTCCCTCGCGCCATCAGGCTACTACACTGACGACA(SEQ ID NO: 36) TGGTTCTACA 454F-BC19-TAG8GCCTCCCTCGCGCCATCAGCTATGCACACTGACGACA (SEQ ID NO: 37) TGGTTCTACA454F-BC20-TAG8 GCCTCCCTCGCGCCATCAGGCTATGACACTGACGACA (SEQ ID NO: 38)TGGTTCTACA 454F-BC21-TAG8 GCCTCCCTCGCGCCATCAGCGTGCAACACTGACGACA(SEQ ID NO: 39) TGGTTCTACA 454F-BC22-TAG8GCCTCCCTCGCGCCATCAGATAGCTACACTGACGACA (SEQ ID NO: 40) TGGTTCTACA454F-BC23-TAG8 GCCTCCCTCGCGCCATCAGTGTAGCACACTGACGACA (SEQ ID NO: 41)TGGTTCTACA 454F-BC24-TAG8 GCCTCCCTCGCGCCATCAGGTGCTAACACTGACGACA(SEQ ID NO: 42) TGGTTCTACA 454F-BC25-TAG8GCCTCCCTCGCGCCATCAGGTCATGACACTGACGACA (SEQ ID NO: 43) TGGTTCTACA454F-BC26-TAG8 GCCTCCCTCGCGCCATCAGATCGTGACACTGACGACA (SEQ ID NO: 44)TGGTTCTACA 454F-BC27-TAG8 GCCTCCCTCGCGCCATCAGTGTACGACACTGACGACA(SEQ ID NO: 45) TGGTTCTACA 454F-BC28-TAG8GCCTCCCTCGCGCCATCAGAGTGTAACACTGACGACA (SEQ ID NO: 46) GGTTCTACA454F-BC29-TAG8 GCCTCCCTCGCGCCATCAGTGACAGACACTGACGACA (SEQ ID NO: 47)TGGTTCTACA 454F-BC30-TAG8 GCCTCCCTCGCGCCATCAGGATCACACACTGACGACA(SEQ ID NO: 48) TGGTTCTACA 454F-BC31-TAG8GCCTCCCTCGCGCCATCAGCTAGAGACACTGACGACA (SEQ ID NO: 49) TGGTTCTACA454F-BC32-TAG8 GCCTCCCTCGCGCCATCAGCTAGTCACACTGACGACA (SEQ ID NO: 50)TGGTTCTACA 454F-BC33-TAG8 GCCTCCCTCGCGCCATCAGAGCTAGACACTGACGACA(SEQ ID NO: 51) TGGTTCTACA 454F-BC34-TAG8GCCTCCCTCGCGCCATCAGTGACTGACACTGACGACA (SEQ ID NO: 52) TGGTTCTACA454F-BC35-TAG8 GCCTCCCTCGCGCCATCAGTGATAGACACTGACGACA (SEQ ID NO: 53)TGGTTCTACA 454F-BC36-TAG8 GCCTCCCTCGCGCCATCAGCGTATCACACTGACGACA(SEQ ID NO: 54) TGGTTCTACA 454F-BC37-TAG8GCCTCCCTCGCGCCATCAGGTCTGAACACTGACGACA (SEQ ID NO: 55) TGGTTCTACA454F-BC38-TAG8 GCCTCCCTCGCGCCATCAGCATGACACACTGACGACA (SEQ ID NO: 56)TGGTTCTACA 454F-BC39-TAG8 GCCTCCCTCGCGCCATCAGCGATGAACACTGACGACA(SEQ ID NO: 57) TGGTTCTACA 454F-BC40-TAG8GCCTCCCTCGCGCCATCAGGCTGATACACTGACGACA (SEQ ID NO: 58) TGGTTCTACA454F-BC41-TAG8 GCCTCCCTCGCGCCATCAGCAGTACACACTGACGACA (SEQ ID NO: 59)TGGTTCTACA 454F-BC42-TAG8 GCCTCCCTCGCGCCATCAGGCGACTACACTGACGACA(SEQ ID NO: 60) TGGTTCTACA 454F-BC43-TAG8GCCTCCCTCGCGCCATCAGGTACGAACACTGACGACA (SEQ ID NO: 61) TGGTTCTACA454F-BC44-TAG8 GCCTCCCTCGCGCCATCAGACGCTAACACTGACGACA (SEQ ID NO: 62)TGGTTCTACA 454F-BC45-TAG8 GCCTCCCTCGCGCCATCAGAGCATCACACTGACGACA(SEQ ID NO: 63) TGGTTCTACA 454F-BC46-TAG8GCCTCCCTCGCGCCATCAGGATGCTACACTGACGACA (SEQ ID NO: 64) TGGTTCTACA454F-BC47-TAG8 GCCTCCCTCGCGCCATCAGGTCTGCACACTGACGACA (SEQ ID NO: 65)TGGTTCTACA 454F-BC48-TAG8 GCCTCCCTCGCGCCATCAGATGCGAACACTGACGACA(SEQ ID NO: 66) TGGTTCTACA

TABLE 4 Sequence Name Sequence SEQ ID TAG8-P53-1+ACACTGACGACATGGTTCTACAACTGTCCAGCTTTGT (SEQ ID NO: 67) GCC TAG8-P53-2+ACACTGACGACATGGTTCTACAGATCATCATAGGAGT (SEQ ID NO: 68) TGCATTGTTGTAG8-P53-3+ ACACTGACGACATGGTTCTACACGGACCTTTGTCCTT (SEQ ID NO: 69) CCTTAG8-P53-4+ ACACTGACGACATGGTTCTACAATGCAAACCTCAATC (SEQ ID NO: 70) CCTCCTAG8-P53-5+ ACACTGACGACATGGTTCTACAAGTTTCTTCCCATGC (SEQ ID NO: 71) ACCTGTAG8-P53-6+ ACACTGACGACATGGTTCTACAGTGAATCCCCGTCTC (SEQ ID NO: 72)TACTAAAA TAG8-P53-7+ ACACTGACGACATGGTTCTACATGTTTCCCATTTGCG(SEQ ID NO: 73) GTTATGA TAG8-P53-8+ACACTGACGACATGGTTCTACAAGTTGTGGGACTGCT (SEQ ID NO: 74) TTATACATT454R-P53-1- GCCTTGCCAGCCCGCTCAGTCCTCTGCCTAGGCGTT (SEQ ID NO: 75)454R-P53-2- GCCTTGCCAGCCCGCTCAGGAAATGTAAATGTGGAGC (SEQ ID NO: 76) CAAACA454R-P53-3- GCCTTGCCAGCCCGCTCAGACTCATTCTTGAAAATAC (SEQ ID NO: 77) CTCCGG454R-P53-4- GCCTTGCCAGCCCGCTCAGAAATGCCACCTCGATTTA (SEQ ID NO: 78) GGAAA454R-P53-5- GCCTTGCCAGCCCGCTCAGTCACCCTCCCGAATAGCT (SEQ ID NO: 79)GCCTTGCCAGCCCGCTCAGAGTGTAAAATGGTACAAC (SEQ ID NO: 80) 454R-P53-6- CGCT454R-P53-7- GCCTTGCCAGCCCGCTCAGCCTCTTAAGATACTGTAA (SEQ ID NO: 81)ACTCTGTAAAGC 454R-P53-8- GCCTTGCCAGCCCGCTCAGATTGTGCCATTGTACTCT(SEQ ID NO: 82) AGCC TAG8-P53-9+ ACACTGACGACATGGTTCTACACTTCCTTTCTCTACTG(SEQ ID NO: 83) AATGCTTTTAATTT TAG8-P53-10+ACACTGACGACATGGTTCTACATCTTACACAAACTCT (SEQ ID NO: 84) TCAGAAAACAGATAG8-P53-11+ ACACTGACGACATGGTTCTACAGTACCAAAACCAAA (SEQ ID NO: 85)CAAGGACAT TAG8-P53-12+ ACACTGACGACATGGTTCTACAGGTGAAACGCCATCT(SEQ ID NO: 86) CTACTAA TAG8-P53-13+ACACTGACGACATGGTTCTACATCATGATTGTAGCTG (SEQ ID NO: 87) ATTCAACATTCATAG8-P53-14+ ACACTGACGACATGGTTCTACAACTAGCATGCTGAAA (SEQ ID NO: 88) CCCCTAG8-P53-15+ ACACTGACGACATGGTTCTACATCAGGAGATCGAGA (SEQ ID NO: 89) CCATCCTAG8-P53-16+ ACACTGACGACATGGTTCTACATCATGCCTGTAATCC (SEQ ID NO: 90) CAGC454R-P53-9- GCCTTGCCAGCCCGCTCAGACCTCAAATGATCCCCTG (SEQ ID NO: 91) C454R-P53-10- GCCTTGCCAGCCCGCTCAGATTACAGGCGTGAGCCAC (SEQ ID NO: 92)454R-P53-11- GCCTTGCCAGCCCGCTCAGTTTTGAGATGAAGTCTTG (SEQ ID NO: 93)CTCTGT 454R-P53-12- GCCTTGCCAGCCCGCTCAGTAAAGACCAGTCTGACTA(SEQ ID NO: 94) TGTTGC 454R-P53-13-GCCTTGCCAGCCCGCTCAGACCATGCCCGGCTAATTT (SEQ ID NO: 95) T 454R-P53-14-GCCTTGCCAGCCCGCTCAGAGTTCACGCCATTCTCCT (SEQ ID NO: 96) G 454R-P53-15-GCCTTGCCAGCCCGCTCAGCACTACGCCCGGCTAATT (SEQ ID NO: 97) TT 454R-P53-16-GCCTTGCCAGCCCGCTCAGTGGCCCCATTAGGACATG (SEQ ID NO: 98) TAT TAG8-P53-17+ACACTGACGACATGGTTCTACATTGTCCCATTGCACT (SEQ ID NO: 99) CCAG TAG8-P53-18+ACACTGACGACATGGTTCTACATGGGCAACAAGAGT (SEQ ID NO: 100) GAAACTTAG8-P53-19+ ACACTGACGACATGGTTCTACAAAATAAATATAGCA (SEQ ID NO: 101)GGGTTGCAGGT TAG8-P53-20+ ACACTGACGACATGGTTCTACATGCATTTCTCTTGGC(SEQ ID NO: 102) TCCC TAG8-P53-21+ ACACTGACGACATGGTTCTACAACTTTCCTCAACTCT(SEQ ID NO: 103) ACATTTCCC TAG8-P53-22+ACACTGACGACATGGTTCTACATCAGTGCAAACAACA (SEQ ID NO: 104) GAAAAGTGTAG8-P53-23+ ACACTGACGACATGGTTCTACACATGTTTCTTAGCAA (SEQ ID NO: 105)ATCTGATGACA TAG8-P53-24+ ACACTGACGACATGGTTCTACATCTGTGGTCCCAGCT(SEQ ID NO: 106) ACT 454R-P53-17- GCCTTGCCAGCCCGCTCAGTTTCACCATGTTAGGTTG(SEQ ID NO: 107) GTCTC 454R-P53-18-GCCTTGCCAGCCCGCTCAGTGTAGGTTAAATCCAAAT (SEQ ID NO: 108) ACTATACCGTC454R-P53-19- GCCTTGCCAGCCCGCTCAGTCTCAAATCTTCAGTAGC (SEQ ID NO: 109)AACTAAAATCT 454R-P53-20- GCCTTGCCAGCCCGCTCAGTCCCGACCTCAGGTGATC(SEQ ID NO: 110) 454R-P53-21- GCCTTGCCAGCCCGCTCAGTGGTCTTGAACTCCCAAC(SEQ ID NO: 111) TTC 454R-P53-22- GCCTTGCCAGCCCGCTCAGCCTCCGACTCCCAAAGTG(SEQ ID NO: 112) 454R-P53-23- GCCTTGCCAGCCCGCTCAGACTACAGCCTCGGACTCC(SEQ ID NO: 113) GCCTTGCCAGCCCGCTCAGATCTTGCACGAAGTTATG (SEQ ID NO: 114)454R-P53-24- CAACTA TAG8-P53-25+ ACACTGACGACATGGTTCTACAACCACTGCACTCCAG(SEQ ID NO: 115) C TAG8-P53-26+ ACACTGACGACATGGTTCTACAACAAGGAAAAGTAT(SEQ ID NO: 116) CAGACAATGTAAGT TAG8-P53-27+ACACTGACGACATGGTTCTACAACGGTAGCTCACACC (SEQ ID NO: 117) TGTAATTAG8-P53-28+ ACACTGACGACATGGTTCTACATGGAAGTCCCTCTCT (SEQ ID NO: 118)GATTGT TAG8-P53-29+ ACACTGACGACATGGTTCTACAACTGACTTTCTGCTC(SEQ ID NO: 119) TTGTCTTTC TAG8-P53-30+ACACTGACGACATGGTTCTACAATTCTGGGACAGCCA (SEQ ID NO: 120) AGTC TAG8-P53-31+ACACTGACGACATGGTTCTACAAGGAGTTCAAGACC (SEQ ID NO: 121) AGCCT TAG8-P53-32+ACACTGACGACATGGTTCTACATCTGTCTCCTTCCTCT (SEQ ID NO: 122) TCCTAC454R-P53-25- GCCTTGCCAGCCCGCTCAGCCTCTTCCCCAAAAGCTC (SEQ ID NO: 123) T454R-P53-26- GCCTTGCCAGCCCGCTCAGTCTCGAACTCCTTACTTC (SEQ ID NO: 124) AGGT454R-P53-27- GCCTTGCCAGCCCGCTCAGCCCAACACCATGCCAGTG (SEQ ID NO: 125)454R-P53-28- GCCTTGCCAGCCCGCTCAGTCCCCAGCCCTCCAG (SEQ ID NO: 126)454R-P53-29- GCCTTGCCAGCCCGCTCAGATTGAAGTCTCATGGAAG (SEQ ID NO: 127) CCAG454R-P53-30- GCCTTGCCAGCCCGCTCAGTCAAGTGATCTTCCCACC (SEQ ID NO: 128) TCA454R-P53-31- GCCTTGCCAGCCCGCTCAGACAACCTCCGTCATGTGC (SEQ ID NO: 129)454R-P53-32- GCCTTGCCAGCCCGCTCAGACCCATTTACTTTGCACA (SEQ ID NO: 130)TCTCA TAG8-P53-33+ ACACTGACGACATGGTTCTACATTAAGGGTGGTTGTC(SEQ ID NO: 131) AGTGG TAG8-P53-34+ACACTGACGACATGGTTCTACATTGCAGTGAGCTGAG (SEQ ID NO: 132) ATCACTAG8-P53-35+ ACACTGACGACATGGTTCTACAATCTCCTTACTGCTC (SEQ ID NO: 133)CCACT TAG8-P53-36+ ACACTGACGACATGGTTCTACATTTTATCACCTTTCCT(SEQ ID NO: 134) TGCCTCTT TAG8-P53-37+ACACTGACGACATGGTTCTACAACTCGTCGTAAGTTG (SEQ ID NO: 135) AAAATATTGTAAGTTAG8-P53-38+ ACACTGACGACATGGTTCTACATCCCAAAGTGCTGGG (SEQ ID NO: 136)ATTAC TAG8-P53-39+ ACACTGACGACATGGTTCTACATCCATCCTCCCAGCT(SEQ ID NO: 137) CAG TAG8-P53-40+ ACACTGACGACATGGTTCTACAATCTCAGCTCACTGC(SEQ ID NO: 138) AGC 454R-P53-33- GCCTTGCCAGCCCGCTCAGAGCCAACCTAGGAGATA(SEQ ID NO: 139) ACACA 454R-P53-34-GCCTTGCCAGCCCGCTCAGAGGCTCCATCTACTCCCA (SEQ ID NO: 140) A 454R-P53-35-GCCTTGCCAGCCCGCTCAGTTGATAAGAGGTCCCAAG (SEQ ID NO: 141) ACTTAGTA454R-P53-36- GCCTTGCCAGCCCGCTCAGTGGGTGACAGAGTGAGA (SEQ ID NO: 142) CT454R-P53-37- GCCTTGCCAGCCCGCTCAGACATCACTGTAATCCAGC (SEQ ID NO: 143) CTG454R-P53-38- GCCTTGCCAGCCCGCTCAGAGATCATGCCACTGCACT (SEQ ID NO: 144) C454R-P53-39- GCCTTGCCAGCCCGCTCAGGGCATGTGCCTGTAGTCC (SEQ ID NO: 145)454R-P53-40- GCCTTGCCAGCCCGCTCAGTGGTCTTGAACTCCTGAC (SEQ ID NO: 146) CTTAG8-P53-41+ ACACTGACGACATGGTTCTACAAAACAGCATGGTTGC (SEQ ID NO: 147)ATGAAAG TAG8-P53-42+ ACACTGACGACATGGTTCTACAAGTCGCATGCACATG(SEQ ID NO: 148) TAGTC TAG8-P53-43+ACACTGACGACATGGTTCTACAAAAAGTCAGCTGTAT (SEQ ID NO: 149) AGGTACTTGAAGTAG8-P53-44+ ACACTGACGACATGGTTCTACACCTCAGTGTATCCAC (SEQ ID NO: 150)AGAACA TAG8-P53-45+ ACACTGACGACATGGTTCTACAATGCATGCCTGTAAT(SEQ ID NO: 151) CCCAG TAG8-P53-46+ACACTGACGACATGGTTCTACAAACTCATGTTCAAGA (SEQ ID NO: 152) CAGAAGGGTAG8-P53-47+ ACACTGACGACATGGTTCTACAATTTTCTCTAACTTC (SEQ ID NO: 153)AAGGCCCATAT TAG8-P53-48+ ACACTGACGACATGGTTCTACATGGATCCACCAAGAC(SEQ ID NO: 154) TTGTTTTAT 454R-P53-41-GCCTTGCCAGCCCGCTCAGGATTACAGGTGTGAGCCA (SEQ ID NO: 155) CT 454R-P53-42-GCCTTGCCAGCCCGCTCAGACAGTACCTGAGTTAAAA (SEQ ID NO: 156) GATGGTTC454R-P53-43- GCCTTGCCAGCCCGCTCAGTGAGACCCTCCAGCTCTG (SEQ ID NO: 157)454R-P53-44- GCCTTGCCAGCCCGCTCAGATCTTCCCTTACCCCATTT (SEQ ID NO: 158)TACTTTATT 454R-P53-45- GCCTTGCCAGCCCGCTCAGTTCAAAGACCCAAAACCC(SEQ ID NO: 159) AAAATG 454R-P53-46-GCCTTGCCAGCCCGCTCAGGTCAAGTTCTAGACCCCA (SEQ ID NO: 160) TGTAATA454R-P53-47- GCCTTGCCAGCCCGCTCAGTGTGGTCCCAGCTACTCC (SEQ ID NO: 161)454R-P53-48- GCCTTGCCAGCCCGCTCAGAGCAAAGTTTTATTGTAA (SEQ ID NO: 162)AATAAGAGATCGAT

Example 4 4-Primer Barcoding of Target Nucleic Acids in Preparation for454 DNA Sequencing Using a Microfluidic Device that Permits Recovery ofAmplication Products

Target-specific primers were designed for 48 genomic regions associatedwith prostate cancer. In addition to the target-specific regions, theprimers were designed to contain additional tag sequences at the 5′ end.Forward primers contained the sequence ACACTGACGACATGGTTCTACA (SEQ IDNO:163). Reverse primers contained the sequence TACGGTAGCAGAGACTTGGTCT(SEQ ID NO:164). The sequences of the primers containing both tagsequences and the target-specific regions are listed in Table 5.

TABLE 5 Amplicon Assay Tagged Forward Tagged Reverse Amplicon size (noAssay # Name primer sequence primer sequence position tags)  1 MSMB-1ACACTGACGACAT TACGGTAGCAGA chr10:51219512 + 157 GGTTCTACAGTGGGACTTGGTCTGCA 51219668 TTGCCCTCTCCAG CACGCATATTAAA TA (SEQ ID NO: 547)ATAGGAA (SEQ ID NO: 165)  2 MSMB-2 ACACTGACGACAT TACGGTAGCAGAchr10:51225703 + 208 GGTTCTACATCAT GACTTGGTCTTTC 25125910 TCTCCACCCTGACATCTGCAGACAG CTT (SEQ ID GTCCA NO: 548) (SEQ ID NO: 166)  3 MSMB-3ACACTGACGACAT TACGGTAGCAGA chr10:51226702 + 183 GGTTCTACAAGGCGACTTGGTCTCCA 51226884 CTTGTTCTCATTG GCACTGGCTTGAG CAT (SEQ ID ACTTNO: 549) (SEQ ID NO: 167)  4 MSMB-4 ACACTGACGACAT TACGGTAGCAGAchr10:51232232 + 229 GGTTCTACAGGGT GACTTGGTCTAGG 51232460 CCTTTCTCTTCTACCAGAGGAGAAT ACAGG (SEQ ID GAGG NO: 550) (SEQ ID NO: 168)  5 HNF1B-1ACACTGACGACAT TACGGTAGCAGA chr17:33121423 + 138 GGTTCTACACAGAGACTTGGTCTATG 33121560 GGGTGATGGTGTG ACCCTGCCAAATG GA (SEQ ID ACACNO: 551) (SEQ ID NO: 169)  6 HNF1B-5 ACACTGACGACAT TACGGTAGCAGAchr17:33138980 + 252 GGTTCTACATGCT GACTTGGTCTTGG 33139231 TCCCATTCTTCTTAAACTGCTCTTTG CTCC (SEQ ID TGGTC NO: 552) (SEQ ID NO: 170)  7 HNF1B-6ACACTGACGACAT TACGGTAGCAGA chr17:33144574 + 254 GGTTCTACATGCCGACTTGGTCTTGG 33144827 TCTTATCTTATCA TGGCACTAATGTT GCTCCA (SEQ ID CCCTANO: 553) (SEQ ID NO: 171)  8 HNF1B-7 ACACTGACGACAT TACGGTAGCAGAchr17:33165634 + 234 GGTTCTACATAAG GACTTGGTCTGAG 33165867 ATCCGTGGCAAGGTCCGTGTCTACA AACC (SEQ ID ACTGG NO: 554) (SEQ ID NO: 172)  9 HNF1B-8ACACTGACGACAT TACGGTAGCAGA chr17:33165796 + 175 GGTTCTACAGTCCGACTTGGTCTCCC 33165970 ATGGCCAGCTTTT CTCACTCACCATC G (SEQ ID NO: 555)TCC (SEQ ID NO: 173) 10 HNF1B-9 ACACTGACGACAT TACGGTAGCAGAchr17:33167605 + 215 GGTTCTACAAGGG GACTTGGTCTAGT 13367819 TTCCTGGGTCTGTCCGATGATGCCTG GTA (SEQ ID CT NO: 556) (SEQ ID NO: 174) 11 HNF1B-10ACACTGACGACAT TACGGTAGCAGA chr17:33167782 + 194 GGTTCTACACTTCGACTTGGTCTTGA 33167975 TTGTTGGTGGGCT GTGAAGGCTACA CAG (SEQ ID GACCCTANO: 557) (SEQ ID NO: 175) 12 HNF1B-11 ACACTGACGACAT TACGGTAGCAGAchr17:33173490 + 192 GGTTCTACATGAG GACTTGGTCTAGA 33173681 AGGGCAAAGGTCGGGAGGTGGTCG ACTT (SEQ ID ATGT NO: 558) (SEQ ID NO: 176) 13 HNF1B-12ACACTGACGACAT TACGGTAGCAGA chr17:33173623 + 160 GGTTCTACAGTTGGACTTGGTCTTCT 33173782 AGATGCTGGGAG CCCACTAGTACCC AGGT (SEQ ID TAACCATCNO: 559) (SEQ ID NO: 177) 14 MYC-1 ACACTGACGACAT TACGGTAGCAGAchr8:128817980 + 142 GGTTCTACAGACC GACTTGGTCTGCA 128818121 CGCTTCTCTGAAATTCGACTCATCTC GG (SEQ ID AGCA NO: 560) (SEQ ID NO: 178) 15 MYC-2ACACTGACGACAT TACGGTAGCAGA chr8:128819612 + 247 GGTTCTACACAGGGACTTGGTCTCAG 128819858 TTTCCGCACCAAG CAGCTCGAATTTC A (SEQ ID NO: 561)TTCC (SEQ ID NO: 179) 16 MYC-6 ACACTGACGACAT TACGGTAGCAGAchr8:128821784 + 255 GGTTCTACAAACC GACTTGGTCTCCT 128822038 TTGCTAAAGGAGTCTTGGCAGCAGG GATTTCT (SEQ ID ATAGT NO: 562) (SEQ ID NO: 180) 17 MYC-7ACACTGACGACAT TACGGTAGCAGA chr8:128821968 + 250 GGTTCTACAACGTGACTTGGTCTAAC 128822217 CTCCACACATCAG TCCGGGATCTGGT CAC (SEQ ID CACNO: 563) (SEQ ID NO: 181) 18 MYC-8 ACACTGACGACAT TACGGTAGCAGAchr8:128822158 + 263 GGTTCTACACCAG GACTTGGTCTTTC 128822420 AGGAGGAACGAGTGTTAGAAGGAAT CTAA (SEQ ID CGTTTTCC NO: 564) (SEQ ID NO: 182) 19 JAZF1-2ACACTGACGACAT TACGGTAGCAGA chr7:27846803 + 244 GGTTCTACATTCCGACTTGGTCTCTC 27847046 ATGTGGTTATGCC CTGACAGTCCTTG AAG (SEQ ID CACTTNO: 565) (SEQ ID NO: 183) 20 JAZF1-4 ACACTGACGACAT TACGGTAGCAGAchr7:27998002 + 195 GGTTCTACACAAT GACTTGGTCTCTT 27998196 AAGCAGCAGATATGTGTTAGGTAGC TAAGGTTGTT CTCATATATTC (SEQ ID NO: 566) (SEQ ID NO: 184)21 NCOA4-1 ACACTGACGACAT TACGGTAGCAGA chr10:51249073 + 265 GGTTCTACATTCAGACTTGGTCTGCC 51249337 AAGGTGGTTTTTG CTGTGTCAAGAGT GTTG (SEQ ID CCAGNO: 567) (SEQ ID NO: 185) 22 NCOA4-2 ACACTGACGACAT TACGGTAGCAGAchr10:51250503 + 246 GGTTCTACATTGG GACTTGGTCTACC 51250748 GAAACATCATTCTAGAAGCCATGCTC TTGG (SEQ ID AAAC NO: 568) (SEQ ID NO: 186) 23 NCOA4-3ACACTGACGACAT TACGGTAGCAGA chr10:51250847 + 250 GGTTCTACATGGTGACTTGGTCTTGA 51251096 GTCATTGTGGCTA TCTTATCCTAGCA GTTG (SEQ IDACACAGAAG NO: 569) (SEQ ID NO: 187) 24 NCOA4-4 ACACTGACGACATTACGGTAGCAGA chr10:51251218 + 201 GGTTCTACATGAA GACTTGGTCTAGA 51251418GTTGATGAAACA AGTGCCCAGTGA GATATTCCTT AGCAT (SEQ ID NO: 570)(SEQ ID NO: 188) 25 NCOA4-5 ACACTGACGACAT TACGGTAGCAGA chr10:51252141 +197 GGTTCTACATTGG GACTTGGTCTCCC 51252337 CAGCATAGCATA AAAGGAAGTATAAATAACA (SEQ ID AGCCAAG NO: 571) (SEQ ID NO: 189) 26 NCOA4-6ACACTGACGACAT TACGGTAGCAGA chr10:51252768 + 227 GGTTCTACACTGCGACTTGGTCTTCC 51252994 ATTTGACATTCCT ACCTACTGCTGTG TGTTT (SEQ ID TCTACTGNO: 572) (SEQ ID NO: 190) 27 NCOA4-7 ACACTGACGACAT TACGGTAGCAGAchr10:51254556 + 260 GGTTCTACAGCAG GACTTGGTCTTCT 51254815 ACAGAATCTCCAAGATAGGTCCATCT AGCA (SEQ ID CATCTTGA NO: 573) (SEQ ID NO: 191) 28 NCOA4-8ACACTGACGACAT TACGGTAGCAGA chr10:51254768 + 255 GGTTCTACAGGTTGACTTGGTCTTGG 51255022 GGAGATCAAGAG TCATTCAGGCACT CTTCC (SEQ ID TCAGNO: 574) (SEQ ID NO: 192) 29 NCOA4-9 ACACTGACGACAT TACGGTAGCAGAchr10:51254962 + 253 GGTTCTACAGAAA GACTTGGTCTCCT 51255214 CCAGCCCAAAGGTCTTTCTTCAGAA TGT (SEQ ID GCCACT NO: 575) (SEQ ID NO: 193) 30 NCOA4-10ACACTGACGACAT TACGGTAGCAGA chr10:51255167 + 266 GGTTCTACAGAATGACTTGGTCTTGG 51255432 TGTGAGAAGGAG GACTTCCTTCTTT GCTCTG (SEQ ID GTATGGNO: 576) (SEQ ID NO: 194) 31 NCOA4-11 ACACTGACGACAT TACGGTAGCAGAchr10:51255385 + 249 GGTTCTACACCTT GACTTGGTCTCCA 51255633 GTCGGAGTGGCTTGTGCTATTTTGAT ATC (SEQ ID GTTTATGC NO: 577) (SEQ ID NO: 195) 32 NCOA4-13ACACTGACGACAT TACGGTAGCAGA chr10:51259156 + 155 GGTTCTACAGGAGGACTTGGTCTTTG 51259310 CTTTAAGGCAGGG GCAAGCTGCAGTC AAA (SEQ ID ACNO: 578) (SEQ ID NO: 196) 33 NUDT11-1 ACACTGACGACAT TACGGTAGCAGAchrX:51255496 + 253 GGTTCTACAAGCG GACTTGGTCTGTA 51255748 AGGCAGACAAATCTGACTGTCACGG AGAAG (SEQ ID AGCTG NO: 579) (SEQ ID NO: 197) 34 SLC22A3-4ACACTGACGACAT TACGGTAGCAGA chr6:160738955 + 209 GGTTCTACATCTGGACTTGGTCTTCC 160739163 CATTCTGGCATGT CCGTATTAATGCA CTC (SEQ ID TGGTATNO: 580) (SEQ ID NO: 198) 35 SLC22A3-5 ACACTGACGACAT TACGGTAGCAGAchr6:160748030 + 245 GGTTCTACAAAGG GACTTGGTCTTTG 160748274 TGAGCTCTTTTCCTTGGCTATCTGGC TGTCTT (SEQ ID CCTA NO: 581) (SEQ ID NO: 199) 36 SLC22A3-6ACACTGACGACAT TACGGTAGCAGA chr6:160749740 + 268 GGTTCTACATGCTGACTTGGTCTGTC 160750007 TCTGTGACCTCTT TGTTTGGAGTCTA GTGT (SEQ IDATTTCTGC NO: 82) (SEQ ID NO: 200) 37 SLC22A3-7 ACACTGACGACATTACGGTAGCAGA chr6:160751720 + 201 GGTTCTACACATA GACTTGGTCTAAT 160751920ACTCACAACAGC CAATTCACCAGCT CTCCTTC (SEQ ID TTAGCAA NO: 583)(SEQ ID NO: 201) 38 SLC22A3-10 ACACTGACGACAT TACGGTAGCAGAchr6:160778107 + 202 GGTTCTACAGTGG GACTTGGTCTGGC 160778308 TGGAACTGCCAGTCCCTATACTTGA GA (SEQ ID TTGTGG NO: 584) (SEQ ID NO: 202) 39 SLC22A3-11ACACTGACGACAT TACGGTAGCAGA chr6:160783754 + 189 GGTTCTACACCTCGACTTGGTCTCGC 160783942 CCTTTCAAACTTT TGGTCTACAGAGT CTGTG (SEQ IDTACTTAGGA NO: 585) (SEQ ID NO: 203) 40 SLC22A3-12 ACACTGACGACATTACGGTAGCAGA chr6:160784591 + 208 GGTTCTACATGAT GACTTGGTCTTGA 160784798TATCTTGAAGTCA AGGCTCTTAAGAA CTTGTTGAA (SEQ TAGCAAATG ID NO: 586)(SEQ ID NO: 204) 41 SLC22A3-13 ACACTGACGACAT TACGGTAGCAGAchr6:160788700 + 235 GGTTCTACAGTGT GACTTGGTCTTTC 160788934 CTTCCTGGAGCGGCCTGTGGATATTC TAA (SEQ ID AATTTTCT NO: 587) (SEQ ID NO: 205) 42SLC22A3-14 ACACTGACGACAT TACGGTAGCAGA chr6:160791984 + 169 GGTTCTACATCTTGACTTGGTCTATC 160792152 TCCTAAAGACTTT TCTGCAAGGCACA CTCCTTTG (SEQ IDGCTT NO: 588) (SEQ ID NO: 206) 43 KLK3-1 ACACTGACGACAT TACGGTAGCAGAchr19:56049936 + 205 GGTTCTACAAGTC GACTTGGTCTGGA 56050140 CTGGGGAATGAAAAGAGCCTCAGCT GGTT (SEQ ID TGAC NO: 589) (SEQ ID NO: 207) 44 KLK3-2ACACTGACGACAT TACGGTAGCAGA chr19:56051260 + 256 GGTTCTACAGTTCGACTTGGTCTCCT 56051515 CTCCTGTCAACCC CTGGGACACAGA TGA (SEQ ID CACCTNO: 590) (SEQ ID NO: 208) 45 KLK3-3 ACACTGACGACAT TACGGTAGCAGAchr19:56053051 + 250 GGTTCTACA GACTTGGTCTTTC 56053300 TCCTTATCATCCTACAGCATCCGTGA CGCTCCT (SEQ ID GC NO: 591) (SEQ ID NO: 209) 46 KLK3-4ACACTGACGACAT TACGGTAGCAGA chr19:56053237 + 200 GGTTCTACAACTCGACTTGGTCTCCC 56053436 CAGCCACGACCTC TCAGACCCAGGC AT (SEQ ID NO: 592)ATC (SEQ ID NO: 210) 47 KLK3-5 ACACTGACGACAT TACGGTAGCAGAchr19:56053490 + 240 GGTTCTACAGGTC GACTTGGTCTCCC 56053729 CAGCCCACAACAAGCCCAGAATTA GT (SEQ ID NO: 593) AGGT (SEQ ID NO: 211) 48 KLK3-8ACACTGACGACAT TACGGTAGCAGA chr19:56054924 + 192 GGTTCTACATCTTGACTTGGTCTGGG 56055115 CCAAAGCTGGGA CACATGGTTCACT ACTG (SEQ ID GCNO: 594) (SEQ ID NO: 212)

Preparation of Reaction Mixtures

Primers were synthesized by IDT at 10 nmol scale, and providedresuspended in water at a concentration of 100 uM. The forward andreverse primer for each region in Table 5 were combined in separatewells in a 96-well PCR plate (USA scientific) to a final concentrationof 1 μM of each primer in PCR-quality water (Teknova) containing 0.05%Tween-20.

48 human genomic DNA samples from the HapMap sample collection wereresuspended at 50 ng/μl in low-EDTA TE buffer (Teknova), and preparedfor PCR as follows.

A pre-sample mixture was prepared as follows:

TABLE 6 Volume per Volume for 64 samples Pre-sample mixture sample (μl)(μl) Faststart High Fidelity 0.5 32 reaction Buffer with MgCl₂ DMSO 0.16.4 PCR-Grade Nucleotide 0.1 6.4 Mixture Faststart High-Fidelity 0.053.2 Enzyme Blend (Roche 04 738 292 001) 20x Access Array Loading 0.25 16Reagent (PN: 100-0883) 20x Evagreen (Biotium- 0.25 16 31000) 20x ROX dye(Invitrogen 0.25 16 12223-012) PCR-Grade water 0.5 32 Total 2 128

For each sample, a sample mixture containing forward and reverse barcodeprimers, genomic DNA, and pre-sample mix was prepared in an individualwell in a 96-well PCR plate.

TABLE 7 Sample Mixture Volume (μl) Pre-sample Mixture 2 2 μM forwardbarcode primer 0.5 2 μm reverse barcode primer 0.5 Genomic DNA (50ng/μl) 1 PCR-grade water 1

Each sample was mixed with one pair of barcode primers selected fromTable 8.

TABLE 8 Reverse Forward Reverse barcode Forward barcode barcode primerprimer barcode primer primer (454B-BC#-CS1) SEQ ID NO. (454A-BC#-CS2)SEQ ID NO.  1 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGGCAT (SEQ IDTCAGGCATGCTACGG NO: 165) GCACACTGACGACATGGTTCTAC NO: 166)TAGCAGAGACTTGGT A CT  2 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGCGTA(SEQ ID TCAGCGTACGTACGG NO: 167) CGACACTGACGACATGGTTCTAC NO: 168)TAGCAGAGACTTGGT A CT  3 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGGTCA(SEQ ID TCAGGTCAGCTACGG NO: 169) GCACACTGACGACATGGTTCTAC NO: 170)TAGCAGAGACTTGGT A CT  4 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGAGCT(SEQ ID TCAGAGCTGCTACGG NO: 171) GCACACTGACGACATGGTTCTAC NO: 172)TAGCAGAGACTTGGT A CT  5 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGTGCA(SEQ ID TCAGTGCATCTACGG NO: 173) TCACACTGACGACATGGTTCTAC NO: 174)TAGCAGAGACTTGGT A CT  6 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGCTGA(SEQ ID TCAGCTGATGTACGG NO: 175) TGACACTGACGACATGGTTCTAC NO: 176)TAGCAGAGACTTGGT A CT  7 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGGTAG(SEQ ID TCAGGTAGTCTACGG NO: 177) TCACACTGACGACATGGTTCTAC NO: 178)TAGCAGAGACTTGGT A CT  8 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGGTCG(SEQ ID TCAGGTCGATTACGG NO: 179) ATACACTGACGACATGGTTCTAC NO: 180)TAGCAGAGACTTGGT A CT  9 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGGATA(SEQ ID TCAGGATACGTACGG NO: 181) CGACACTGACGACATGGTTCTAC NO: 182)TAGCAGAGACTTGGT A CT 10 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGTGAT(SEQ ID TCAGTGATGCTACGG NO: 183) GCACACTGACGACATGGTTCTAC NO: 184)TAGCAGAGACTTGGT A CT 11 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGAGCT(SEQ ID TCAGAGCTGATACGG NO: 185) GAACACTGACGACATGGTTCTAC NO: 186)TAGCAGAGACTTGGT A CT 12 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGACTG(SEQ ID TCAGACTGTATACGG NO: 187) TAACACTGACGACATGGTTCTAC NO: 188)TAGCAGAGACTTGGT A CT 13 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGTGCA(SEQ ID TCAGTGCATGTACGG NO: 189) TGACACTGACGACATGGTTCTAC NO: 190)TAGCAGAGACTTGGT A CT 14 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGAGTC(SEQ ID TCAGAGTCTATACGG NO: 191) TAACACTGACGACATGGTTCTAC NO: 192)TAGCAGAGACTTGGT A CT 15 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGTGTC(SEQ ID TCAGTGTCTGTACGGT NO: 193) TGACACTGACGACATGGTTCTAC NO: 194)AGCAGAGACTTGGTC A T 16 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGGCTA(SEQ ID TCAGGCTAGCTACGG NO: 195) GCACACTGACGACATGGTTCTAC NO: 196)TAGCAGAGACTTGGT A CT 17 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGGATA(SEQ ID TCAGGATAGCTACGG NO: 197) GCACACTGACGACATGGTTCTAC NO: 198)TAGCAGAGACTTGGT A CT 18 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGGCTA(SEQ ID TCAGGCTACTTACGG NO: 199) CTACACTGACGACATGGTTCTAC NO: 200)TAGCAGAGACTTGGT A CT 19 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGCTAT(SEQ ID TCAGCTATGCTACGG NO: 201) GCACACTGACGACATGGTTCTAC NO: 202)TAGCAGAGACTTGGT A CT 20 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGGCTA(SEQ ID TCAGGCTATGTACGG NO: 203) TGACACTGACGACATGGTTCTAC NO: 204)TAGCAGAGACTTGGT A CT 21 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGCGTG(SEQ ID TCAGCGTGCATACGG NO: 205) CAACACTGACGACATGGTTCTAC NO: 206)TAGCAGAGACTTGGT A CT 22 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGATAG(SEQ ID TCAGATAGCTTACGG NO: 207) CTACACTGACGACATGGTTCTAC NO: 208)TAGCAGAGACTTGGT A CT 23 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGTGTA(SEQ ID TCAGTGTAGCTACGG NO: 209) GCACACTGACGACATGGTTCTAC NO: 210)TAGCAGAGACTTGGT A CT 24 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGGTGC(SEQ ID TCAGGTGCTATACGG NO: 211) TAACACTGACGACATGGTTCTAC NO: 212)TAGCAGAGACTTGGT A CT 25 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGGTCA(SEQ ID TCAGGTCATGTACGG NO: 213) TGACACTGACGACATGGTTCTAC NO: 214)TAGCAGAGACTTGGT A CT 26 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGATCG(SEQ ID TCAGATCGTGTACGG NO: 215) TGACACTGACGACATGGTTCTAC NO: 216)TAGCAGAGACTTGGT A CT 27 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGTGTA(SEQ ID TCAGTGTACGTACGG NO: 217) CGACACTGACGACATGGTTCTAC NO: 218)TAGCAGAGACTTGGT A CT 28 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGAGTG(SEQ ID TCAGAGTGTATACGG NO: 219) TAACACTGACGACATGGTTCTAC NO: 220)TAGCAGAGACTTGGT A CT 29 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGTGAC(SEQ ID TCAGTGACAGTACGG NO: 221) AGACACTGACGACATGGTTCTAC NO: 222)TAGCAGAGACTTGGT A CT 30 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGGATC(SEQ ID TCAGGATCACTACGG NO: 223) ACACACTGACGACATGGTTCTAC NO: 224)TAGCAGAGACTTGGT A CT 31 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGCTAG(SEQ ID TCAGCTAGAGTACGG NO: 225) AGACACTGACGACATGGTTCTAC NO: 226)TAGCAGAGACTTGGT A CT 32 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGCTAG(SEQ ID TCAGCTAGTCTACGG NO: 227) TCACACTGACGACATGGTTCTAC NO: 228)TAGCAGAGACTTGGT A CT 33 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGAGCT(SEQ ID TCAGAGCTAGTACGG NO: 229) AGACACTGACGACATGGTTCTAC NO: 230)TAGCAGAGACTTGGT A CT 34 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGTGAC(SEQ ID TCAGTGACTGTACGG NO: 231) TGACACTGACGACATGGTTCTAC NO: 232)TAGCAGAGACTTGGT A CT 35 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGTGAT(SEQ ID TCAGTGATAGTACGG NO: 233) AGACACTGACGACATGGTTCTAC NO: 234)TAGCAGAGACTTGGT A CT 36 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGCGTA(SEQ ID TCAGCGTATCTACGG NO: 235) TCACACTGACGACATGGTTCTAC NO: 236)TAGCAGAGACTTGGT A CT 37 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGGTCT(SEQ ID TCAGGTCTGATACGG NO: 237) GAACACTGACGACATGGTTCTAC NO: 238)TAGCAGAGACTTGGT A CT 38 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGCATG(SEQ ID TCAGCATGACTACGG NO: 239) ACACACTGACGACATGGTTCTAC NO: 240)TAGCAGAGACTTGGT A CT 39 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGCGAT(SEQ ID TCAGCGATGATACGG NO: 241) GAACACTGACGACATGGTTCTAC NO: 242)TAGCAGAGACTTGGT A CT 40 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGGCTG(SEQ ID TCAGGCTGATTACGG NO: 243) ATACACTGACGACATGGTTCTAC NO: 244)TAGCAGAGACTTGGT A CT 41 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGCAGT(SEQ ID TCAGCAGTACTACGG NO: 245) ACACACTGACGACATGGTTCTAC NO: 246)TAGCAGAGACTTGGT A CT 42 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGGCGA(SEQ ID TCAGGCGACTTACGG NO: 247) CTACACTGACGACATGGTTCTAC NO: 248)TAGCAGAGACTTGGT A CT 43 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGGTAC(SEQ ID TCAGGTACGATACGG NO: 249) GAACACTGACGACATGGTTCTAC NO: 250)TAGCAGAGACTTGGT A CT 44 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGACGC(SEQ ID TCAGACGCTATACGG NO: 251) TAACACTGACGACATGGTTCTAC NO: 252)TAGCAGAGACTTGGT A CT 45 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGAGCA(SEQ ID TCAGAGCATCTACGG NO: 253) TCACACTGACGACATGGTTCTAC NO: 254)TAGCAGAGACTTGGT A CT 46 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGGATG(SEQ ID TCAGGATGCTTACGG NO: 255) CTACACTGACGACATGGTTCTAC NO: 256)TAGCAGAGACTTGGT A CT 47 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGGTCT(SEQ ID TCAGGTCTGCTACGG NO: 257) GCACACTGACGACATGGTTCTAC NO: 258)TAGCAGAGACTTGGT A CT 48 GCCTTGCCAGCCCGC (SEQ ID GCCTCCCTCGCGCCATCAGATGC(SEQ ID TCAGATGCGATACGG NO: 259) GAACACTGACGACATGGTTCTAC NO: 260)TAGCAGAGACTTGGT A CT

Running the Access Array IFC

The containment and interface accumulator reservoirs were filled with300 μl of Control Line Fluid (Fluidigm PN 89000020) and the H1-H4reagent wells were loaded with 500 μl of 0.05% Tween-20 in PCR-gradewater prior to Access Array IFC loading. 5 μl of each sample mixture wasloaded into the sample ports, and 5 μl of each primer mixture was loadedinto the primer inlets on the Access Array IFC.

The Access Array IFC was thermal cycled and imaged using a BioMark™Real-Time PCR system manufactured by Fluidigm Corporation. The AccessArray IFC thermal cycling protocol contains a thermal mix step [50° C.for 2 min, 70° C. for 20 min], a hotstart step [95° C. for 10 min], a 35cycle touch down PCR strategy [2 cycles of 95° C. for 15 sec and 63° C.for 1 min, 2 cycles of 95° C. for 15 sec and 62° C. for 1 min, 2 cyclesof 95° C. for 15 sec and 61° C. for 1 min, 2 cycles of 95° C. for 15 secand 60° C. for 1 min, 2 cycles of 95° C. for 15 sec and 58° C. for 1min, 25 cycles of 95° C. for 15 sec and 72° C. for 1 min], and anelongation step [72° C. for 3 min]. The real-time data was analyzed withFluidigm Real-Time PCR Analysis software to obtain C_(T) values for eachreaction chamber.

After amplification, the PCR products were harvested from the AccessArray IFC using the Post-PCR IFC Loader AX. Before harvesting, eachsample port was filled with 2 μl of 0.05% Tween-20. Residual solutionwas removed from the H1-H4 reagent wells, and they were refilled with600 μl of 1× Access Array Harvesting Reagent (0.05% tween-20). Afterharvesting, each sample port became a PCR product outlet that contained10 μl (±10%) of 48 pooled PCR products. The pooled PCR products wereremoved from the Access Array IFC and stored in a microtiter plate at 4°C.

1 μl of each PCR product pool for each sample was taken and loaded ontoan Agilent 1K Bioanalyzer chip. FIG. 12 shows the electropherograms fromeach of the 48 individual product pools. FIG. 13 shows the distributionof product size within a single product pool. All products fall withinthe predicted size range, and there is no evidence of any small-sizedPCR by-products.

PCR Products for each sample were pooled based on concentrationscalculated from the Agilent Bioanalyzer traces. The product pool waspurified using AMPure beads (Agencourt) according to the manufacturer'sinstructions.

The purified product pool was subjected to emulsion PCR followed bypyrosequencing on a 454 FLX sequencer (Roche Analytical Sciences)according to manufacturer's instructions. The sequence file output bythe sequencer was then analyzed for the presence of barcoded PCRproducts.

The number of sequences obtained for each barcode were counted, andplotted (FIG. 14(A)). On average ˜3400 sequences were counted for eachbarcode. All samples were represented at >50% of average and <2-fold ofaverage.

The number of sequences counted for each individual PCR product in eachsample were then analyzed (FIG. 14(B)). Only one of 2304 PCR productswas not observed on the sequencer. The vast majority of sequences werepresent at >50% of average and <2 fold of average. 2303/2304 productswere counted >5 times. FIG. 14(C) shows the distribution of PCR productsfrom all 2304 PCR reactions in the Access Array IFC. >95% of sequenceswere measured between 50% and 2 fold of the average coverage. >99% ofsequences were measured between 50% and 2 fold of the average coverage.

Example 5 Multi-Primer Amplification Using Four Outer Primers withDifferent Combinations of Primer Binding Site and Nucleotide Tags

Sets of primer pairs were designed to amplify specific regions from theEGFR and MET genes. These were then combined in an Access Array IFC withhuman genomic DNA and four outer primers (FIG. 15).

Preparation of Reaction Mixtures

Primers were synthesized by Eurofins MWG Operon at 10 nmol scale andprovided resuspended in water at a concentration of 100 μM. The forwardand reverse primer for each region in Table 9 were combined in separatewells in a 96-well PCR plate (USA scientific) to a final concentrationof 1 μM of each primer in PCR-quality water (Teknova) containing 0.05%Tween-20.

TABLE 9 Forward Reverse Primer Primer SEQ ID SEQ ID Assay Forward PrimerNO. Reverse Primer NO. EGFR_Exon3 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACATTCTT NO: 261) TCTCCAGCCTCTCACCCTGNO: 262) AGACCATCCAGGA TAAA GGTG EGFR_Exon4 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACAAGCT NO: 263) TCTTAGGAGCTGGAGGCAGNO: 264) GGAAAGAGTGCTC AGAT ACC EGFR_Exon5 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACAGCGT NO: 265) TCTACATGGGTCTGAGGCTNO: 266) CATCAGTTTCTCAT GTTC CATT EGFR_Exon6 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACACCCT NO: 267) TCTTCTTACCAGGCAGTCGNO: 268) GGGAAATGATCCT CTCT ACC EGFR_Exon7 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACACCAG NO: 269) TCTGACAAGGATGCCTGACNO: 270) CGTGTCCTCTCTCC CAGT T EGFR_Exon8 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACACAAA NO: 271) TCTGATGTGTTCCTTTGGANO: 272) GGAGGATGGAGCC GGTGG TTTC EGFR_Exon9 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACATCCA NO: 273) TCTCAAGCAACTGAACCTGNO: 274) ACAAATGTGAACG TGACTC GAAT EGFR_Exon10 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACAGATC NO: 275) TCTTTCCAAGGGAACAGGANO: 276) AATAATCACCCTG AATATG TTGTTTG EGFR_Exon11 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACATCCTA NO: 277) TCTGCTTTGGCTGTGGTCANO: 278) CGTGGTGTGTGTCT ACTT GA EGFR_Exon12 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACACCAC NO: 279) TCTCGGTGACTTACTGCAGNO: 280) ATGATTTTTCTTCT CTGTT CTCCA EGFR_Exon13 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACAGCTCT NO: 281) TCTGCTATAACAACAACCTNO: 282) GTCACTGACTGCT GGAGCCT GTG EGFR_Exon14 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACAGCTG NO: 283) TCTGACGTGGATAGCAGCANO: 284) ACGGGTTTCCTCTT AGG C EGFR_Exon15 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACAGCAT NO: 285) TCTTTCTGTTCTCCTTCACTNO: 286) GAACATTTTTCTCC TTCCAC ACCT EGFR_Exon16 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACATTTCT NO: 287) TCTCCACAGCAGTGTGGTCNO: 288) CTTTCACTTCCTAC ATTC AGATGC EGFR_Exon17 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACATGGA NO: 289) TCTCCCAGGACTGGCACTCNO: 290) ATCTGTCAGCAAC A CTC EGFR_Exon18 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACAGCTG NO: 291) TCTCCCACCAGACCATGAGNO: 292) AGGTGACCCTTGT AGG CTC EGFR_Exon19 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACATCAC NO: 293) TCTCCACACAGCAAAGCAGNO: 294) AATTGCCAGTTAA AAAC CGTCT EGFR_Exon20 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACACCAC NO: 295) TCTCCGTATCTCCCTTCCCTNO: 296) ACTGACGTGCCTC GAT TC EGFR_Exon21 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACACCTC NO: 297) TCTCTGACCTAAAGCCACCNO: 298) ACAGCAGGGTCTT TCCTT CTC EGFR_Exon22 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACACACT NO: 299) TCTCCAGCTTGGCCTCAGTNO: 300) GCCTCATCTCTCAC ACA CA EGFR_Exon23 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACACATG NO: 301) TCTAGTGTGGACAGACCCANO: 302) ATCCCACTGCCTTC CCA TT EGFR_Exon24 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACATTCCA NO: 303) TCTGAGGGACTCTTCCCAANO: 304) GTGTTCTAATTGCA TGGA CTGTT EGFR_Exon25 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACACTAA NO: 305) TCTTTTGTTCAAATGAGTANO: 306) TAGCCTCAAAATC GACACAGC TCTGCAC EGFR_Exon26 ACACTGACGACAT(SEQ ID TACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACACATTC NO: 307)TCTTTCTGGCTTATAAGGT NO: 308) CATGGGCAACTTC GTTCATACA TC EGFR_Exon27ACACTGACGACAT (SEQ ID TACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACACCTTCNO: 309) TCTTCCAGACAAGCCACTC NO: 310) CCTCATTTCCTCCT ACC G EGFR_Exon8-12ACACTGACGACAT (SEQ ID TACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACAcctctgat NO: 311) TCTCTAATTTGGTGGCTGC NO: 312) ttctttccactttca CTTT EGFR_Exon28-2ACACTGACGACAT (SEQ ID TACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACATGTC NO: 313)TCTGGTCCTGGGTATCGAA NO: 314) AACAGCACATTCG AGAGT ACAG EGFR_Exon2ACACTGACGACAT (SEQ ID TACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACATTTCTNO: 315) TCTAGGAAAATCAAAGTCA NO: 316) TCCAGTTTGCCAA CCAACC GGMET_Exon1-1 ACACTGACGACAT (SEQ ID TACGGTAGCAGAGACTTGG (SEQ IDGGTTCTACACTCTC NO: 317) TCTCAGCACAGGCCCAGTC NO: 318) GCCTTGAACCTGTT TT TMET_Exon1-2 ACACTGACGACAT (SEQ ID TACGGTAGCAGAGACTTGG (SEQ IDGGTTCTACATTCCT NO: 319) TCTGGGAGAATATGCAGTG NO: 320) TGGTGCCACTAACAACCTC TACA MET_Exon2 ACACTGACGACAT (SEQ ID TACGGTAGCAGAGACTTGG (SEQ IDGGTTCTACATGGA NO: 321) TCTTTGCACAATACCAGAT NO: 322) TTCACATTAACTCTAGAACAGAC ATGACCA MET_Exon3 ACACTGACGACAT (SEQ ID TACGGTAGCAGAGACTTGG(SEQ ID GGTTCTACATGAG NO: 323) TCTCGTCTATGGAAATTCC NO: 324)CTTGTTGGAATAA CTGTG GGATG MET_Exon4 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACAGAAG NO: 325) TCTTGCCAGCTGTTAGAGANO: 326) CTCTTTCCACCCCT TTCCT TC MET_Exon5 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACATGTCC NO: 327) TCTCCCCAGCAAAGCATTTNO: 328) TTGTAGGTTTTCCC TAAG AAA MET_Exon6 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACAGAAA NO: 329) TCTCATGATAGGATAGAATNO: 330) ATTCCTTGGATTTG CTTCCTTACCA TCATG MET_Exon7 ACACTGACGACAT(SEQ ID TACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACAGTTTT NO: 331)TCTTTCAAATTGACAGATG NO: 332) GTTTTTATCTCCCC CAACAA TCCA MET_Exon8ACACTGACGACAT (SEQ ID TACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACAGGAA NO: 333)TCTTTGTTTTCTTATACCCA NO: 334) CCATTGAGTTATAT TCAGAAGC CCTTTTG MET_Exon9ACACTGACGACAT (SEQ ID TACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACATTGGTNO: 335) TCTCAGGTACCATGAAAGC NO: 336) GGAAAGAACCTCT CACA CAA MET_Exon10ACACTGACGACAT (SEQ ID TACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACATGTTGNO: 337) TCTTTTGAGCTGATGATTT NO: 338) CCAAGCTGTATTCT AAGACAGTG GTTMET_Exon12 ACACTGACGACAT (SEQ ID TACGGTAGCAGAGACTTGG (SEQ IDGGTTCTACAGGAC NO: 339) TCTCAAGAATCGACGACAA NO: 340) CCAAAGTGCTACATCTTAAAC ACC MET_Exon13 ACACTGACGACAT (SEQ ID TACGGTAGCAGAGACTTGG(SEQ ID GGTTCTACAGCCC NO: 341) TCTCAACAATGTCACAACC NO: 342)ATGATAGCCGTCT CACTG TTA MET_Exon14 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACACCTTC NO: 343) TCTGCTTACTGGAAAATCGNO: 344) ATCTTACAGATCA TATTTAACAAA GTTTCCT MET_Exon15 ACACTGACGACAT(SEQ ID TACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACAACGC NO: 345)TCTTCCACAAGGGGAAAGT NO: 346) AGTGCTAACCAAG GTAAA TTCT MET_Exon16ACACTGACGACAT (SEQ ID TACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACATGTCTNO: 347) TCTGGCTTACAGCTAGTTT NO: 348) CCACCACTGGATT GCCAGT TCTMET_Exon17 ACACTGACGACAT (SEQ ID TACGGTAGCAGAGACTTGG (SEQ IDGGTTCTACATGCTT NO: 349) TCTTCCTCCTTGTCACTTAA NO: 350) TTCTAACTCTCTTTTTTGGA GACTGC MET_Exon18 ACACTGACGACAT (SEQ ID TACGGTAGCAGAGACTTGG(SEQ ID GGTTCTACATTCTA NO: 351) TCTAGAGGAGAAACTCAG NO: 352)TTTCAGCCACGGG AGATAACCAA TAA MET_Exon19 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACACTCA NO: 353) TCTGGCATTTCTGTAAAAGNO: 354) CCTCATCTGTCCTG TAAAGAACG TTTCT MET_Exon20 ACACTGACGACAT (SEQ IDTACGGTAGCAGAGACTTGG (SEQ ID GGTTCTACACCTG NO: 355) TCTGTGTGGACTGTTGCTTNO: 356) CCTTCAAAGGGTC TGACA TCT

A single human Genomic DNA sample (Coriell NA10830) was resuspended at50 ng/μl in low-EDTA TE buffer (Teknova) and prepared for PCR asfollows.

A pre-sample mixture was prepared as follows:

TABLE 10 Volume per Volume for Pre-sample mixture sample (μl) 64 samples(μl) Faststart High Fidelity 0.5 32 reaction Buffer with MgCl₂ DMSO 0.16.4 PCR-Grade Nucleotide 0.1 6.4 Mixture Faststart High-Fidelity 0.053.2 Enzyme Blend (Roche 04 738 292 001) 20x Access Array Loading 0.25 16Reagent (PN: 100-0883) PCR-Grade water 0.5 32 Total 2.5 160

For each sample replicate, a sample mixture containing forward andreverse barcode primers, genomic DNA and pre-sample mix was prepared inan individual well in a 96-well PCR plate.

TABLE 11 Sample Mixture Volume (μl) Pre-sample Mixture 2 4 μM forwardbarcode primer 0.5 4 μm reverse barcode primer 0.5 Genomic DNA (50ng/μl) 1 PCR-grade water 1

Four replicate samples were prepared by mixing each sample with one pairof barcode primers selected from Table 12.

TABLE 12 Reverse Forward Reverse barcode Forward barcode barcode primerprimer barcode primer primer (454B-BC#-CS1) SEQ ID NO. (454A-BC#-CS2)SEQ ID NO.  1 GCCTTGCCAGCCC (SEQ ID NO: 357) GCCTCCCTCGCGCC(SEQ ID NO: 358) GCTCAGGCATGC ATCAGGCATGCAC TACGGTAGCAGA ACTGACGACATGGTGACTTGGTCT TCTACA  2 GCCTTGCCAGCCC (SEQ ID NO: 359) GCCTCCCTCGCGCC(SEQ ID NO: 360) GCTCAGCGTACG ATCAGCGTACGAC TACGGTAGCAGA ACTGACGACATGGTGACTTGGTCT TCTACA  3 GCCTTGCCAGCCC (SEQ ID NO: 361) GCCTCCCTCGCGCC(SEQ ID NO: 362) GCTCAGGTCAGC ATCAGGTCAGCAC TACGGTAGCAGA ACTGACGACATGGTGACTTGGTCT TCTACA  4 GCCTTGCCAGCCC (SEQ ID NO: 363) GCCTCCCTCGCGCC(SEQ ID NO: 364) GCTCAGAGCTGC ATCAGAGCTGCAC TACGGTAGCAGA ACTGACGACATGGTGACTTGGTCT TCTACA  5 GCCTTGCCAGCCC (SEQ ID NO: 365) GCCTCCCTCGCGCC(SEQ ID NO: 366) GCTCAGTGCATCT ATCAGTGCATCACA ACGGTAGCAGAGCTGACGACATGGTT ACTTGGTCT CTACA  6 GCCTTGCCAGCCC (SEQ ID NO: 367)GCCTCCCTCGCGCC (SEQ ID NO: 368) GCTCAGCTGATGT ATCAGCTGATGACAACGGTAGCAGAG CTGACGACATGGTT ACTTGGTCT CTACA  7 GCCTTGCCAGCCC(SEQ ID NO: 369) GCCTCCCTCGCGCC (SEQ ID NO: 370) GCTCAGGTAGTCTATCAGGTAGTCACA ACGGTAGCAGAG CTGACGACATGGTT ACTTGGTCT CTACA  8GCCTTGCCAGCCC (SEQ ID NO: 371) GCCTCCCTCGCGCC (SEQ ID NO: 372)GCTCAGGTCGATT ATCAGGTCGATACA ACGGTAGCAGAG CTGACGACATGGTT ACTTGGTCT CTACA 9 GCCTTGCCAGCCC (SEQ ID NO: 373) GCCTCCCTCGCGCC (SEQ ID NO: 374)GCTCAGGATACG ATCAGGATACGAC TACGGTAGCAGA ACTGACGACATGGT GACTTGGTCT TCTACA10 GCCTTGCCAGCCC (SEQ ID NO: 375) GCCTCCCTCGCGCC (SEQ ID NO: 376)GCTCAGTGATGCT ATCAGTGATGCACA ACGGTAGCAGAG CTGACGACATGGTT ACTTGGTCT CTACA11 GCCTTGCCAGCCC (SEQ ID NO: 377) GCCTCCCTCGCGCC (SEQ ID NO: 378)GCTCAGAGCTGA ATCAGAGCTGAAC TACGGTAGCAGA ACTGACGACATGGT GACTTGGTCT TCTACA12 GCCTTGCCAGCCC (SEQ ID NO: 379) GCCTCCCTCGCGCC (SEQ ID NO: 380)GCTCAGACTGTAT ATCAGACTGTAACA ACGGTAGCAGAG CTGACGACATGGTT ACTTGGTCT CTACA13 GCCTTGCCAGCCC (SEQ ID NO: 381) GCCTCCCTCGCGCC (SEQ ID NO: 382)GCTCAGTGCATGT ATCAGTGCATGACA ACGGTAGCAGAG CTGACGACATGGTT ACTTGGTCT CTACA14 GCCTTGCCAGCCC (SEQ ID NO: 383) GCCTCCCTCGCGCC (SEQ ID NO: 384)GCTCAGAGTCTAT ATCAGAGTCTAACA ACGGTAGCAGAG CTGACGACATGGTT ACTTGGTCT CTACA15 GCCTTGCCAGCCC (SEQ ID NO: 385) GCCTCCCTCGCGCC (SEQ ID NO: 386)GCTCAGTGTCTGT ATCAGTGTCTGACA ACGGTAGCAGAG CTGACGACATGGTT ACTTGGTCT CTACA16 GCCTTGCCAGCCC (SEQ ID NO: 387) GCCTCCCTCGCGCC (SEQ ID NO: 388)GCTCAGGCTAGC ATCAGGCTAGCAC TACGGTAGCAGA ACTGACGACATGGT GACTTGGTCT TCTACA

Running the Access Array IFC

The containment and interface accumulator reservoirs were filled with300 μl of Control Line Fluid (Fluidigm PN 89000020), and the H1-H4reagent wells were loaded with 500 μl of 0.05% Tween-20 in PCR-gradewater prior to Access Array IFC loading. 5 μl of each sample mixture wasloaded into the sample ports, and 5 μl of each primer mixture was loadedinto the primer inlets on the Access Array IFC.

The Access Array IFC was thermal cycled and imaged using an IFCStand-Alone Thermal Cycler (Fluidigm Corporation). The thermal cyclingprotocol contains a thermal mix step [50° C. for 2 min, 70° C. for 20min], a hotstart step [95° C. for 10 min], a 35 cycle PCR strategy [2cycles of 95° C. for 15 sec and 60° C. for 4 min, 33 cycles of 95° C.for 15 sec, 60° C. for 15 sec, 72° C. for 1 min, and an elongation step[72° C. for 3 min].

After amplification, the PCR products were harvested from the AccessArray IFC using the Post-PCR IFC Loader AX. Before harvesting, eachsample port was filled with 2 μl of 0.05% Tween-20. Residual solutionwas removed from the H1-H4 reagent wells, and they were refilled with600 μl of 1× Access Array Harvesting Reagent (0.05% tween-20). Afterharvesting each sample port became a PCR product outlet that contained10 μl (±10%) of 48 pooled PCR products. The pooled PCR products wereremoved from the Access Array IFC and stored in a microtiter plate at 4°C.

PCR products for each sample were pooled based on concentrationscalculated from the Agilent Bioanalyzer traces. The purified productpool was subjected to emulsion PCR followed by pyrosequencing on a 454FLX sequencer (Roche Analytical Sciences) according to manufacturer'sinstructions. Emulsion PCR reactions were run with beads containing bothA and B primer sequences attached, enabling sequence reads for bothstrands of the amplicon.

The number of sequences counted for each individual PCR product in eachsample were analyzed to demonstrate representation of the PCR productsshown in FIG. 15. Sequences could be counted for each of the ampliconsshown in FIG. 15B, by summing tag 5 sequences from emulsion A with tag 8sequences from emulsion B (FIG. 16A) or tag 5 sequences from emulsion Bwith tag 8 sequences from emulsion A. FIG. 16 shows that representationof all amplicons mostly lies between 2× and 0.5× of average coverage.Furthermore, representation of both amplicons for each primer pair isvery similar, although the amplicon represented in FIG. 16B shows lessvariation within samples.

Example 6 4-Primer Barcoding of Target Nucleic Acids for Illumina DNASequencing Using a Microfluidic Device that Permits Recovery ofAmplication Products

Sequences designed for a 4-primer tagging scheme to be used on theIllumina Genome Analyzer II are shown in Tables 13 and 14. The tagsequence is the inner primer sequence.

TABLE 13 Inner primers Target-Specific Tag Sequence Sequence (Forward)Oligonucleotide Sequence ACACTCTTTCCCTACA ACTGTCCAGCTTTACACTCTTTCCCTACACGACGCTCTTCCG CGACGCTCTTCCGAT GTGCCATCTACTGTCCAGCTTTGTGCC CT (SEQ ID NO: 390) (SEQ ID NO: 391)(SEQ ID NO: 389) ACACTCTTTCCCTACA GATCATCATAGGAACACTCTTTCCCTACACGACGCTCTTCCG CGACGCTCTTCCGAT GTTGCATTGTTGATCTGATCATCATAGGAGTTGCATTGTTG CT (SEQ ID NO: 393) (SEQ ID NO: 394)(SEQ ID NO: 392) Target-Specific Tag Sequence Sequence (Reverse)Oligonucleotide Sequence CTCGGCATTCCTGCTG TCCTCTGCCTAGGCTCGGCATTCCTGCTGAACCGCTCTTCCG AACCGCTCTTCCGAT CGTT ATCTTCCTCTGCCTAGGCGTTCT (SEQ ID NO: 396) (SEQ ID NO: 397) (SEQ ID NO: 395) CTCGGCATTCCTGCTGGAAATGTAAATGT CTCGGCATTCCTGCTGAACCGCTCTTCCG AACCGCTCTTCCGAT GGAGCCAAACAATCTGAAATGTAAATGTGGAGCCAAACA CT (SEQ ID NO: 399) (SEQ ID NO: 400)(SEQ ID NO: 398)

TABLE 14 Barcode Primers Direction ILMN_PE1sh_F ForwardAATGATACGGCGACCAC CGAGATC TACACTCTTT CCCTACACGA (SEQ ID NO: 401)ILMN_PE2sh_R Reverse CAAGCAGAAGACGGCATA CGAGATC GGTCTCGG CATTCCTGCTGAAC(SEQ ID NO: 402)

The successful amplication of a PCR product using the 4-primer strategydesigned for use on the Illumina GA II sequencer is shown in FIG. 17.

Example 7 Barcoding of Target Nucleic Acids for Titanium Chemistry onthe 454 FLX Sequencer (Roche Analytical Sciences)

Table 15 shows forward barcode sequences for use with Titanium chemistryon the 454 FLX Sequencer (Roche Analytical Sciences). Table 16 showsreverse barcode sequences for use with Titanium chemistry on the 454 FLXSequencer (Roche Analytical Sciences).

TABLE 15 Forward Well Barcode Oligo Name Forward Oligo sequenceSEQ ID NO. A1 TI-MID1 TI-F-MID1- CGTATCGCCTCCCTCGCGCCATCA(SEQ ID NO: 403) TAG8 GACGAGTGCGTACACTGACGACA TGGTTCTACA B1 TI-MID2TI-F-MID2- CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 404) TAG8GACGCTCGACAACACTGACGACA TGGTTCTACA C1 TI-MID3 TI-F-MID3-CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 405) TAG8 GAGACGCACTCACACTGACGACATGGTTCTACA D1 TI-MID67 TI-F-MID67- CGTATCGCCTCCCTCGCGCCATCA(SEQ ID NO: 406) TAG8 GTCGATAGTGAACACTGACGACA TGGTTCTACA E1 TI-MID5TI-F-MID5- CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 407) TAG8GATCAGACACGACACTGACGACA TGGTTCTACA F1 TI-MID6 TI-F-MID6-CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 408) TAG8 GATATCGCGAGACACTGACGACATGGTTCTACA G1 TI-MID7 TI-F-MID7- CGTATCGCCTCCCTCGCGCCATCA(SEQ ID NO: 409) TAG8 GCGTGTCTCTAACACTGACGACAT GGTTCTACA H1 TI-MID8TI-F-MID8- CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 410) TAG8GCTCGCGTGTCACACTGACGACAT GGTTCTACA A2 TI-MID10 TI-F-MID10-CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 411) TAG8 GTCTCTATGCGACACTGACGACATGGTTCTACA B2 TI-MID11 TI-F-MID11- CGTATCGCCTCCCTCGCGCCATCA(SEQ ID NO: 412) TAG8 GTGATACGTCTACACTGACGACAT GGTTCTACA C2 TI-MID13TI-F-MID13- CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 413) TAG8GCATAGTAGTGACACTGACGACA TGGTTCTACA D2 TI-MID14 TI-F-MID14-CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 414) TAG8 GCGAGAGATACACACTGACGACATGGTTCTACA E2 TI-MID15 TI-F-MID15- CGTATCGCCTCCCTCGCGCCATCA(SEQ ID NO: 415) TAG8 GATACGACGTAACACTGACGACA TGGTTCTACA F2 TI-MID16TI-F-MID16- CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 416) TAG8GTCACGTACTAACACTGACGACAT GGTTCTACA G2 TI-MID17 TI-F-MID17-CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 417) TAG8 GCGTCTAGTACACACTGACGACATGGTTCTACA H2 TI-MID18 TI-F-MID18- CGTATCGCCTCCCTCGCGCCATCA(SEQ ID NO: 418) TAG8 GTCTACGTAGCACACTGACGACAT GGTTCTACA A3 TI-MID19TI-F-MID19- CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 419) TAG8GTGTACTACTCACACTGACGACAT GGTTCTACA B3 TI-MID20 TI-F-MID20-CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 420) TAG8 GACGACTACAGACACTGACGACATGGTTCTACA C3 TI-MID21 TI-F-MID21- CGTATCGCCTCCCTCGCGCCATCA(SEQ ID NO: 421) TAG8 GCGTAGACTAGACACTGACGACA TGGTTCTACA D3 TI-MID22TI-F-MID22- CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 422) TAG8GTACGAGTATGACACTGACGACA TGGTTCTACA E3 TI-MID23 TI-F-MID23-CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 423) TAG8 GTACTCTCGTGACACTGACGACATGGTTCTACA F3 TI-MID24 TI-F-MID24- CGTATCGCCTCCCTCGCGCCATCA(SEQ ID NO: 424) TAG8 GTAGAGACGAGACACTGACGACA TGGTTCTACA G3 TI-MID25TI-F-MID25- CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 425) TAG8GTCGTCGCTCGACACTGACGACAT GGTTCTACA H3 TI-MID26 TI-F-MID26-CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 426) TAG8 GACATACGCGTACACTGACGACATGGTTCTACA A4 TI-MID27 TI-F-MID27- CGTATCGCCTCCCTCGCGCCATCA(SEQ ID NO: 427) TAG8 GACGCGAGTATACACTGACGACA TGGTTCTACA B4 TI-MID28TI-F-MID28- CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 428) TAG8GACTACTATGTACACTGACGACAT GGTTCTACA C4 TI-MID68 TI-F-MID68-CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 429) TAG8 GTCGCTGCGTAACACTGACGACATGGTTCTACA D4 TI-MID30 TI-F-MID30- CGTATCGCCTCCCTCGCGCCATCA(SEQ ID NO: 430) TAG8 GAGACTATACTACACTGACGACA TGGTTCTACA E4 TI-MID31TI-F-MID31- CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 431) TAG8GAGCGTCGTCTACACTGACGACAT GGTTCTACA F4 TI-MID32 TI-F-MID32-CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 432) TAG8 GAGTACGCTATACACTGACGACATGGTTCTACA G4 TI-MID33 TI-F-MID33- CGTATCGCCTCCCTCGCGCCATCA(SEQ ID NO: 433) TAG8 GATAGAGTACTACACTGACGACA TGGTTCTACA H4 TI-MID34TI-F-MID34- CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 434) TAG8GCACGCTACGTACACTGACGACA TGGTTCTACA A5 TI-MID35 TI-F-MID35-CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 435) TAG8 GCAGTAGACGTACACTGACGACATGGTTCTACA B5 TI-MID36 TI-F-MID36- CGTATCGCCTCCCTCGCGCCATCA(SEQ ID NO: 436) TAG8 GCGACGTGACTACACTGACGACA TGGTTCTACA C5 TI-MID37TI-F-MID37- CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 437) TAG8GTACACACACTACACTGACGACA TGGTTCTACA D5 TI-MID38 TI-F-MID38-CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 438) TAG8 GTACACGTGATACACTGACGACATGGTTCTACA E5 TI-MID39 TI-F-MID39- CGTATCGCCTCCCTCGCGCCATCA(SEQ ID NO: 439) TAG8 GTACAGATCGTACACTGACGACA TGGTTCTACA F5 TI-MID40TI-F-MID40- CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 440) TAG8GTACGCTGTCTACACTGACGACAT GGTTCTACA G5 TI-MID69 TI-F-MID69-CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 441) TAG8 GTCTGACGTCAACACTGACGACATGGTTCTACA H5 TI-MID42 TI-F-MID42- CGTATCGCCTCCCTCGCGCCATCA(SEQ ID NO: 442) TAG8 GTCGATCACGTACACTGACGACAT GGTTCTACA A6 TI-MID43TI-F-MID43- CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 443) TAG8GTCGCACTAGTACACTGACGACAT GGTTCTACA B6 TI-MID44 TI-F-MID44-CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 444) TAG8 GTCTAGCGACTACACTGACGACATGGTTCTACA C6 TI-MID45 TI-F-MID45- CGTATCGCCTCCCTCGCGCCATCA(SEQ ID NO: 445) TAG8 GTCTATACTATACACTGACGACAT GGTTCTACA D6 TI-MID46TI-F-MID46- CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 446) TAG8GTGACGTATGTACACTGACGACAT GGTTCTACA E6 TI-MID47 TI-F-MID47-CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 447) TAG8 GTGTGAGTAGTACACTGACGACATGGTTCTACA F6 TI-MID48 TI-F-MID48- CGTATCGCCTCCCTCGCGCCATCA(SEQ ID NO: 448) TAG8 GACAGTATATAACACTGACGACA TGGTTCTACA G6 TI-MID49TI-F-MID49- CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 449) TAG8GACGCGATCGAACACTGACGACA TGGTTCTACA H6 TI-MID50 TI-F-MID50-CGTATCGCCTCCCTCGCGCCATCA (SEQ ID NO: 450) TAG8 GACTAGCAGTAACACTGACGACATGGTTCTACA

TABLE 16 Reverse Oligo Well Barcode Name Reverse Oligo SequenceSEQ ID NO. A1 TI-MID1 TI-R-MID1- CTATGCGCCTTGCCAGCCCGCTCA(SEQ ID NO: 451) TAG5 GACGAGTGCGTTACGGTAGCAGA GACTTGGTCT B1 TI-MID2TI-R-MID2- CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 452) TAG5GACGCTCGACATACGGTAGCAGA GACTTGGTCT C1 TI-MID3 TI-R-MID3-CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 453) TAG5 GAGACGCACTCTACGGTAGCAGAGACTTGGTCT D1 TI-MID67 TI-R-MID67- CTATGCGCCTTGCCAGCCCGCTCA(SEQ ID NO: 454) TAG5 GTCGATAGTGATACGGTAGCAGA GACTTGGTCT E1 TI-MID5TI-R-MID5- CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 455) TAG5GATCAGACACGTACGGTAGCAGA GACTTGGTCT F1 TI-MID6 TI-R-MID6-CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 456) TAG5 GATATCGCGAGTACGGTAGCAGAGACTTGGTCT G1 TI-MID7 TI-R-MID7- CTATGCGCCTTGCCAGCCCGCTCA(SEQ ID NO: 457) TAG5 GCGTGTCTCTATACGGTAGCAGAG ACTTGGTCT H1 TI-MID8TI-R-MID8- CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 458) TAG5GCTCGCGTGTCTACGGTAGCAGA GACTTGGTCT A2 TI-MID10 TI-R-MID10-CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 459) TAG5 GTCTCTATGCGTACGGTAGCAGAGACTTGGTCT B2 TI-MID11 TI-R-MID11- CTATGCGCCTTGCCAGCCCGCTCA(SEQ ID NO: 460) TAG5 GTGATACGTCTTACGGTAGCAGA GACTTGGTCT C2 TI-MID13TI-R-MID13- CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 461) TAG5GCATAGTAGTGTACGGTAGCAGA GACTTGGTCT D2 TI-MID14 TI-R-MID14-CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 462) TAG5 GCGAGAGATACTACGGTAGCAGAGACTTGGTCT E2 TI-MID15 TI-R-MID15- CTATGCGCCTTGCCAGCCCGCTCA(SEQ ID NO: 463) TAG5 GATACGACGTATACGGTAGCAGA GACTTGGTCT F2 TI-MID16TI-R-MID16- CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 464) TAG5GTCACGTACTATACGGTAGCAGA GACTTGGTCT G2 TI-MID17 TI-R-MID17-CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 465) TAG5 GCGTCTAGTACTACGGTAGCAGAGACTTGGTCT H2 TI-MID18 TI-R-MID18- CTATGCGCCTTGCCAGCCCGCTCA(SEQ ID NO: 466) TAG5 GTCTACGTAGCTACGGTAGCAGA GACTTGGTCT A3 TI-MID19TI-R-MID19- CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 467) TAG5GTGTACTACTCTACGGTAGCAGAG ACTTGGTCT B3 TI-MID20 TI-R-MID20-CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 468) TAG5 GACGACTACAGTACGGTAGCAGAGACTTGGTCT C3 TI-MID21 TI-R-MID21- CTATGCGCCTTGCCAGCCCGCTCA(SEQ ID NO: 469) TAG5 GCGTAGACTAGTACGGTAGCAGA GACTTGGTCT D3 TI-MID22TI-R-MID22- CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 470) TAG5GTACGAGTATGTACGGTAGCAGA GACTTGGTCT E3 TI-MID23 TI-R-MID23-CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 471) TAG5 GTACTCTCGTGTACGGTAGCAGAGACTTGGTCT F3 TI-MID24 TI-R-MID24- CTATGCGCCTTGCCAGCCCGCTCA(SEQ ID NO: 472) TAG5 GTAGAGACGAGTACGGTAGCAGA GACTTGGTCT G3 TI-MID25TI-R-MID25- CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 473) TAG5GTCGTCGCTCGTACGGTAGCAGA GACTTGGTCT H3 TI-MID26 TI-R-MID26-CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 474) TAG5 GACATACGCGTTACGGTAGCAGAGACTTGGTCT A4 TI-MID27 TI-R-MID27- CTATGCGCCTTGCCAGCCCGCTCA(SEQ ID NO: 475) TAG5 GACGCGAGTATTACGGTAGCAGA GACTTGGTCT B4 TI-MID28TI-R-MID28- CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 476) TAG5GACTACTATGTTACGGTAGCAGA GACTTGGTCT C4 TI-MID68 TI-R-MID68-CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 477) TAG5 GTCGCTGCGTATACGGTAGCAGAGACTTGGTCT D4 TI-MID30 TI-R-MID30- CTATGCGCCTTGCCAGCCCGCTCA(SEQ ID NO: 478) TAG5 GAGACTATACTTACGGTAGCAGA GACTTGGTCT E4 TI-MID31TI-R-MID31- CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 479) TAG5GAGCGTCGTCTTACGGTAGCAGA GACTTGGTCT F4 TI-MID32 TI-R-MID32-CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 480) TAG5 GAGTACGCTATTACGGTAGCAGAGACTTGGTCT G4 TI-MID33 TI-R-MID33- CTATGCGCCTTGCCAGCCCGCTCA(SEQ ID NO: 481) TAG5 GATAGAGTACTTACGGTAGCAGA GACTTGGTCT H4 TI-MID34TI-R-MID34- CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 482) TAG5GCACGCTACGTTACGGTAGCAGA GACTTGGTCT A5 TI-MID35 TI-R-MID35-CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 483) TAG5 GCAGTAGACGTTACGGTAGCAGAGACTTGGTCT B5 TI-MID36 TI-R-MID36- CTATGCGCCTTGCCAGCCCGCTCA(SEQ ID NO: 484) TAG5 GCGACGTGACTTACGGTAGCAGA GACTTGGTCT C5 TI-MID37TI-R-MID37- CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 485) TAG5GTACACACACTTACGGTAGCAGA GACTTGGTCT D5 TI-MID38 TI-R-MID38-CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 486) TAG5 GTACACGTGATTACGGTAGCAGAGACTTGGTCT E5 TI-MID39 TI-R-MID39- CTATGCGCCTTGCCAGCCCGCTCA(SEQ ID NO: 487) TAG5 GTACAGATCGTTACGGTAGCAGA GACTTGGTCT F5 TI-MID40TI-R-MID40- CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 488) TAG5GTACGCTGTCTTACGGTAGCAGAG ACTTGGTCT G5 TI-MID69 TI-R-MID69-CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 489) TAG5 GTCTGACGTCATACGGTAGCAGAGACTTGGTCT H5 TI-MID42 TI-R-MID42- CTATGCGCCTTGCCAGCCCGCTCA(SEQ ID NO: 490) TAG5 GTCGATCACGTTACGGTAGCAGA GACTTGGTCT A6 TI-MID43TI-R-MID43- CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 491) TAG5GTCGCACTAGTTACGGTAGCAGA GACTTGGTCT B6 TI-MID44 TI-R-MID44-CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 492) TAG5 GTCTAGCGACTTACGGTAGCAGAGACTTGGTCT C6 TI-MID45 TI-R-MID45- CTATGCGCCTTGCCAGCCCGCTCA(SEQ ID NO: 493) TAG5 GTCTATACTATTACGGTAGCAGAG ACTTGGTCT D6 TI-MID46TI-R-MID46- CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 494) TAG5GTGACGTATGTTACGGTAGCAGA GACTTGGTCT E6 TI-MID47 TI-R-MID47-CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 495) TAG5 GTGTGAGTAGTTACGGTAGCAGAGACTTGGTCT F6 TI-MID48 TI-R-MID48- CTATGCGCCTTGCCAGCCCGCTCA(SEQ ID NO: 496) TAG5 GACAGTATATATACGGTAGCAGA GACTTGGTCT G6 TI-MID49TI-R-MID49- CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 497) TAG5GACGCGATCGATACGGTAGCAGA GACTTGGTCT H6 TI-MID50 TI-R-MID50-CTATGCGCCTTGCCAGCCCGCTCA (SEQ ID NO: 498) TAG5 GACTAGCAGTATACGGTAGCAGAGACTTGGTCT

Example 8 Multiplex Barcoding of Target Nucleic Acids

Three pools of 10 primers were assembled from the primers listed inTable 9. PCR conditions were identical to those listed in Example 4,with the exception that primer concentrations were varied. FIG. 18 showsthe results of PCR reactions of three pools of 10 sets of PCR primers(A, B, C) when the PCR reactions were run for template-specific primersonly and in 4-primer mode. The presence of higher molecular weightproducts in the 4-primer strategy demonstrates successful 4-primerassembly.

1-65. (canceled)
 66. A method of operating a microfluidic device, saiddevice comprising independent sample or first reagent inputs combinedwith independent assay or second reagent inputs in an M×N arrayconfiguration, the method comprising: loading M samples or firstreagents into sample chambers through sample input lines arranged ascolumns; loading N assays or second reagents into assay chambers throughassay input lines arranged as rows crossing the columns; wherein duringloading, the sample chambers and the assay chambers are in fluidicisolation; opening an interface valve between pairs of M sample and Nassay chambers to provide fluid communication between M×N pairwisecombinations of M samples or first reagents and N assays or secondreagents, such that each M sample or first reagent is combined with eachof N assay or second reagent to produce M×N unique pairwise reactions;reacting the M×N unique pairwise reactions to form reaction products;flowing a harvesting reagent from a harvesting port to the samplechambers of each column to recover reaction products from themicrofluidic device in separate reaction pools, each reaction poolcontaining a given M sample or first reagent reacted with each of the Nassays or second reagents.
 67. The method of claim 66 further comprisingthermocycling the M×N unique pairwise reactions.
 68. The method of claim66 wherein recovering the reaction products from the microfluidic devicecomprises flowing the reaction products through at least a portion ofthe sample input lines to sample input ports.
 69. The method of claim 68wherein flowing the reaction products through at least a portion of thesample input lines comprises performing dilation pumping.
 70. The methodof claim 69 wherein dilation pumping comprises a fluid flow rate of lessthan or equal to 10 μl per hour.
 71. The method of claim 69 whereinperforming dilation pumping comprises: a) closing a first valve disposedbetween the sample chamber and the sample input port; b) opening asecond valve disposed between the harvesting port and the samplechamber; c) closing the second valve; d) opening the first valve; andrepeating steps (a) through (d) a predetermined number of times.
 72. Themethod of claim 70 wherein the fluid flow rate is less than or equal to5 μl per hour.
 73. The method of claim 72 wherein the fluid flow rate isless than or equal to 1 μl per hour.
 74. The method of claim 66 whereinproduction of M×N unique pairwise reactions comprises a free interfacediffusion process.
 75. The method of claim 66 wherein removing thereaction product comprises a fluid flow rate of less than or equal to 10microliters per hour.
 76. The method of claim 75 wherein the fluid flowrate is less than or equal to 5 microliters per hour.
 77. The method ofclaim 76 wherein the fluid flow rate is less than or equal to 2microliters per hour.
 78. The method of claim 77 wherein the fluid flowrate is less than or equal to 1 microliters per hour.
 79. The method ofclaim 66 wherein recovering the reaction products comprises removing atleast 95% of the reaction products from the microfluidic device. 80-95.(canceled)